Erythropoietin: remodeling and glycoconjugation of erythropoietin

ABSTRACT

The invention includes methods and compositions for remodeling a peptide molecule, including the addition or deletion of one or more glycosyl groups to a peptide, and/or the addition of a modifying group to a peptide.

BACKGROUND OF THE INVENTION

Most naturally occurring peptides contain carbohydrate moieties attachedto the peptide via specific linkages to a select number of amino acidsalong the length of the primary peptide chain. Thus, many naturallyoccurring peptides are termed “glycopeptides.” The variability of theglycosylation pattern on any given peptide has enormous implications forthe function of that peptide. For example, the structure of the N-linkedglycans on a peptide can impact various characteristics of the peptide,including the protease susceptibility, intracellular trafficking,secretion, tissue targeting, biological half-life and antigenicity ofthe peptide in a cell or organism. The alteration of one or more ofthese characteristics greatly affects the efficacy of a peptide in itsnatural setting, and also affects the efficacy of the peptide as atherapeutic agent in situations where the peptide has been generated forthat purpose.

The carbohydrate structure attached to the peptide chain is known as a“glycan” molecule. The specific glycan structure present on a peptideaffects the solubility and aggregation characteristics of the peptide,the folding of the primary peptide chain and therefore its functional orenzymatic activity, the resistance of the peptide to proteolytic attackand the control of proteolysis leading to the conversion of inactiveforms of the peptide to active forms. Importantly, terminal sialic acidresidues present on the glycan molecule affect the length of the halflife of the peptide in the mammalian circulatory system. Peptides whoseglycans do not contain terminal sialic acid residues are rapidly removedfrom the circulation by the liver, an event which negates any potentialtherapeutic benefit of the peptide.

The glycan structures found in naturally occurring glycopeptides aretypically divided into two classes, N-linked and O-linked glycans.

Peptides expressed in eukaryotic cells are typically N-glycosylated onasparagine residues at sites in the peptide primary structure containingthe sequence asparagine-X-serine/threonine where X can be any amino acidexcept proline and aspartic acid. The carbohydrate portion of suchpeptides is known as an N-linked glycan. The early events ofN-glycosylation occur in the endoplasmic reticulum (ER) and areidentical in mammals, plants, insects and other higher eukaryotes.First, an oligosaccharide chain comprising fourteen sugar residues isconstructed on a lipid carrier molecule. As the nascent peptide istranslated and translocated into the ER, the entire oligosaccharidechain is transferred to the amide group of the asparagine residue in areaction catalyzed by a membrane bound glycosyltransferase enzyme. TheN-linked glycan is further processed both in the ER and in the Golgiapparatus. The further processing generally entails removal of some ofthe sugar residues and addition of other sugar residues in reactionscatalyzed by glycosidases and glycosyltransferases specific for thesugar residues removed and added.

Typically, the final structures of the N-linked glycans are dependentupon the organism in which the peptide is produced. For example, ingeneral, peptides produced in bacteria are completely unglycosylated.Peptides expressed in insect cells contain high mannose andpaunci-mannose N-linked oligosaccharide chains, among others. Peptidesproduced in mammalian cell culture are usually glycosylated differentlydepending, e.g., upon the species and cell culture conditions. Even inthe same species and under the same conditions, a certain amount ofheterogeneity in the glycosyl chains is sometimes encountered. Further,peptides produced in plant cells comprise glycan structures that differsignificantly from those produced in animal cells. The dilemma in theart of the production of recombinant peptides, particularly when thepeptides are to be used as therapeutic agents, is to be able to generatepeptides that are correctly glycosylated, i.e., to be able to generate apeptide having a glycan structure that resembles, or is identical tothat present on the naturally occurring form of the peptide. Mostpeptides produced by recombinant means comprise glycan structures thatare different from the naturally occurring glycans.

A variety of methods have been proposed in the art to customize theglycosylation pattern of a peptide including those described in WO99/22764, WO 98/58964, WO 99/54342 and U.S. Pat. No. 5,047,335, amongothers. Essentially, many of the enzymes required for the in vitroglycosylation of peptides have been cloned and sequenced. In someinstances, these enzymes have been used in vitro to add specific sugarsto an incomplete glycan molecule on a peptide. In other instances, cellshave been genetically engineered to express a combination of enzymes anddesired peptides such that addition of a desired sugar moiety to anexpressed peptide occurs within the cell.

Peptides may also be modified by addition of O-linked glycans, alsocalled mucin-type glycans because of their prevalence on mucinousglycopeptide. Unlike N-glycans that are linked to asparagine residuesand are formed by en bloc transfer of oligosaccharide from lipid-boundintermediates, O-glycans are linked primarily to serine and threonineresidues and are formed by the stepwise addition of sugars fromnucleotide sugars (Tanner et al., Biochim. Biophys. Acta. 906:81-91(1987); and Hounsell et al., Glycoconj. J. 13:19-26 (1996)). Peptidefunction can be affected by the structure of the O-linked glycanspresent thereon. For example, the activity of P-selectin ligand isaffected by the O-linked glycan structure present thereon. For a reviewof O-linked glycan structures, see Schachter and Brockhausen, TheBiosynthesis of Branched O-Linked Glycans, 1989, Society forExperimental Biology, pp. 1-26 (Great Britain). Other glycosylationpatterns are formed by linking glycosylphosphatidylinositol to thecarboxyl-terminal carboxyl group of the protein (Takeda et al., TrendsBiochem. Sci. 20:367-371 (1995); and Udenfriend et al., Ann. Rev.Biochem. 64:593-591 (1995).

Although various techniques currently exist to modify the N-linkedglycans of peptides, there exists in the art the need for a generallyapplicable method of producing peptides having a desired, i.e., acustomized glycosylation pattern. There is a particular need in the artfor the customized in vitro glycosylation of peptides, where theresulting peptide can be produced at industrial scale. This and otherneeds are met by the present invention.

The administration of glycosylated and non-glycosylated peptides forengendering a particular physiological response is well known in themedicinal arts. Among the best known peptides utilized for this purposeis insulin, which is used to treat diabetes. Enzymes have also been usedfor their therapeutic benefits. A major factor, which has limited theuse of therapeutic peptides is the immunogenic nature of most peptides.In a patient, an immunogenic response to an administered peptide canneutralize the peptide and/or lead to the development of an allergicresponse in the patient. Other deficiencies of therapeutic peptidesinclude suboptimal potency and rapid clearance rates. The problemsinherent in peptide therapeutics are recognized in the art, and variousmethods of eliminating the problems have been investigated. To providesoluble peptide therapeutics, synthetic polymers have been attached tothe peptide backbone.

Poly(ethylene glycol) (“PEG”) is an exemplary polymer that has beenconjugated to peptides. The use of PEG to derivatize peptidetherapeutics has been demonstrated to reduce the immunogenicity of thepeptides and prolong the clearance time from the circulation. Forexample, U.S. Pat. No. 4,179,337 (Davis et al.) concerns non-immunogenicpeptides, such as enzymes and peptide hormones coupled to polyethyleneglycol (PEG) or polypropylene glycol. Between 10 and 100 moles ofpolymer are used per mole peptide and at least 15% of the physiologicalactivity is maintained.

WO 93/15189 (Veronese et al.) concerns a method to maintain the activityof polyethylene glycol-modified proteolytic enzymes by linking theproteolytic enzyme to a macromolecularized inhibitor. The conjugates areintended for medical applications.

The principal mode of attachment of PEG, and its derivatives, topeptides is a non-specific bonding through a peptide amino acid residue.For example, U.S. Pat. No. 4,088,538 discloses an enzymatically activepolymer-enzyme conjugate of an enzyme covalently linked to PEG.Similarly, U.S. Pat. No. 4,496,689 discloses a covalently attachedcomplex of α-1 protease inhibitor with a polymer such as PEG ormethoxypoly(ethylene glycol) (“mPEG”). Abuchowski et al. (J. Biol. Chem.252: 3578 (1977) discloses the covalent attachment of mPEG to an aminegroup of bovine serum albumin. U.S. Pat. No. 4,414,147 discloses amethod of rendering interferon less hydrophobic by conjugating it to ananhydride of a dicarboxylic acid, such as poly(ethylene succinicanhydride). PCT WO 87/00056 discloses conjugation of PEG andpoly(oxyethylated) polyols to such proteins as interferon-β,interleukin-2 and immunotoxins. EP 154,316 discloses and claimschemically modified lymphokines, such as IL-2 containing PEG bondeddirectly to at least one primary amino group of the lymphokine. U.S.Pat. No. 4,055,635 discloses pharmaceutical compositions of awater-soluble complex of a proteolytic enzyme linked covalently to apolymeric substance such as a polysaccharide.

Another mode of attaching PEG to peptides is through the non-specificoxidation of glycosyl residues on a peptide. The oxidized sugar isutilized as a locus for attaching a PEG moiety to the peptide. Forexample, M'Timkulu (WO 94/05332) discloses the use of a hydrazine- oramino-PEG to add PEG to a glycoprotein. The glycosyl moieties arerandomly oxidized to the corresponding aldehydes, which are subsequentlycoupled to the amino-PEG. See also, Bona et al. (WO 96/40731), where aPEG is added to an inmuunoglobulin molecule by enzymatically oxidizing aglycan on the innmunoglobulin and then contacting the glycan with anamino-PEG molecule.

In each of the methods described above, poly(ethylene glycol) is addedin a random, non-specific manner to reactive residues on a peptidebackbone. For the production of therapeutic peptides, it is clearlydesirable to utilize a derivatization strategy that results in theformation of a specifically labeled, readily characterizable,essentially homogeneous product.

Two principal classes of enzymes are used in the synthesis ofcarbohydrates, glycosyltransferases (e.g., sialyltransferases,oligosaccharyltransferases, N-acetylglucosaminyltransferases), andglycosidases. The glycosidases are further classified as exoglycosidases(e.g., β-mannosidase, β-glucosidase), and endoglycosidases (e.g.,Endo-A, Endo-M). Each of these classes of enzymes has been successfullyused synthetically to prepare carbohydrates. For a general review, see,Crout et al., Curr. Opin. Chem. Biol. 2: 98-111 (1998).

Glycosyltransferases modify the oligosaccharide structures on peptides.Glycosyltransferases are effective for producing specific products withgood stereochemical and regiochemical control. Glycosyltransferases havebeen used to prepare oligosaccharides and to modify terminal N- andO-linked carbohydrate structures, particularly on peptides produced inmammalian cells. For example, the terminal oligosaccharides ofglycopeptides have been completely sialylated and/or fucosylated toprovide more consistent sugar structures, which improves glycopeptidepharmacodynamics and a variety of other biological properties. Forexample, β-1,4-galactosyltransferase is used to synthesize lactosamine,an illustration of the utility of glycosyltransferases in the synthesisof carbohydrates (see, e.g., Wong et al., J. Org. Chem. 47: 5416-5418(1982)). Moreover, numerous synthetic procedures have made use ofα-sialyltransferases to transfer sialic acid fromcytidine-5′-monophospho-N-acetylneuraminic acid to the 3-OH or 6-OH ofgalactose (see, e.g., Kevin et al., Chem. Eur. J. 2: 1359-1362 (1996)).Fucosyltransferases are used in synthetic pathways to transfer a fucoseunit from guanosine-5′-diphosphofucose to a specific hydroxyl of asaccharide acceptor. For example, Ichikawa prepared sialyl Lewis-X by amethod that involves the fucosylation of sialylated lactosamine with acloned fucosyltransferase (Ichikawa et al., J. Am. Chem. Soc. 114:9283-9298 (1992)). For a discussion of recent advances in glycoconjugatesynthesis for therapeutic use see, Koeller et al., Nature Biotechnology18: 835-841 (2000). See also, U.S. Pat. Nos. 5,876,980; 6,030,815;5,728,554; 5,922,577; and WO/9831826.

Glycosidases can also be used to prepare saccharides. Glycosidasesnormally catalyze the hydrolysis of a glycosidic bond. However, underappropriate conditions, they can be used to form this linkage. Mostglycosidases used for carbohydrate synthesis are exoglycosidases; theglycosyl transfer occurs at the non-reducing terminus of the substrate.The glycosidase binds a glycosyl donor in a glycosyl-enzyme intermediatethat is either intercepted by water to yield the hydrolysis product, orby an acceptor, to generate a new glycoside or oligosaccharide. Anexemplary pathway using an exoglycosidase is the synthesis of the coretrisaccharide of all N-linked glycopeptides, including the β-mannosidelinkage, which is formed by the action of β-mannosidase (Singh et al.,Chem. Commun. 993-994 (1996)).

In another exemplary application of the use of a glycosidase to form aglycosidic linkage, a mutant glycosidase has been prepared in which thenormal nucleophilic amino acid within the active site is changed to anon-nucleophilic amino acid. The mutant enzyme does not hydrolyzeglycosidic linkages, but can still form them. Such a mutant glycosidaseis used to prepare oligosaccharides using an α-glycosyl fluoride donorand a glycoside acceptor molecule (Withers et al., U.S. Pat. No.5,716,812).

Although their use is less common than that of the exoglycosidases,endoglycosidases are also utilized to prepare carbohydrates. Methodsbased on the use of endoglycosidases have the advantage that anoligosaccharide, rather than a monosaccharide, is transferred.Oligosaccharide fragments have been added to substrates usingendo-β-N-acetylglucosamines such as endo-F, eizdo-M (Wang et al.,Tetrahedron Lett. 37: 1975-1978); and Haneda et al., Carbohydr. Res.292: 61-70 (1996)).

In addition to their use in preparing carbohydrates, the enzymesdiscussed above are applied to the synthesis of glycopeptides as well.The synthesis of a homogenous glycoform of ribonuclease B has beenpublished (Witte K. et al., J. Am. Chem. Soc. 119: 2114-2118 (1997)).The high mannose core of ribonuclease B was cleaved by treating theglycopeptide with endoglycosidase H. The cleavage occurred specificallybetween the two core GlcNAc residues. The tetrasaccharide sialyl Lewis Xwas then enzymatically rebuilt on the remaining GlcNAc anchor site onthe now homogenous protein by the sequential use ofβ-1,4-galactosyltransferase, α-2,3-sialyltransferase andα-1,3-fucosyltransferase V. However, while each enzymatically catalyzedstep proceeded in excellent yield, such procedures have not been adaptedfor the generation of glycopeptides on an industrial scale.

Methods combining both chemical and enzymatic synthetic elements arealso known in the art. For example, Yamamoto and coworkers (Carbohydr.Res. 305: 415-422 (1998)) reported the chemoenzymatic synthesis of theglycopeptide, glycosylated Peptide T, using an endoglycosidase. TheN-acetylglucosaminyl peptide was synthesized by purely chemical means.The peptide was subsequently enzymatically elaborated with theoligosaccharide of human transferrin peptide. The saccharide portion wasadded to the peptide by treating it with anendo-β-N-acetylglucosaminidase. The resulting glycosylated peptide washighly stable and resistant to proteolysis when compared to the peptideT and N-acetylglucosaminyl peptide T.

The use of glycosyltransferases to modify peptide structure withreporter groups has been explored. For example, Brossmer et al. (U.S.Pat. No. 5,405,753) discloses the formation of a fluorescent-labeledcytidine monophosphate (“CMP”) derivative of sialic acid and the use ofthe fluorescent glycoside in an assay for sialyl transferase activityand for the fluorescent-labeling of cell surfaces, glycoproteins andpeptides. Gross et al. (Analyt. Biochem. 186: 127 (1990)) describe asimilar assay. Bean et al. (U.S. Pat. No. 5,432,059) discloses an assayfor glycosylation deficiency disorders utilizing reglycosylation of adeficiently glycosylated protein. The deficient protein isreglycosylated with a fluorescent-labeled CMP glycoside. Each of thefluorescent sialic acid derivatives is substituted with the fluorescentmoiety at either the 9-position or at the amine that is normallyacetylated in sialic acid. The methods using the fluorescent sialic acidderivatives are assays for the presence of glycosyltransferases or fornon-glycosylated or improperly glycosylated glycoproteins. The assaysare conducted on small amounts of enzyme or glycoprotein in a sample ofbiological origin. The enzymatic derivatization of a glycosylated ornon-glycosylated peptide on a preparative or industrial scale using amodified sialic acid has not been disclosed or suggested in the priorart.

Considerable effort has also been directed towards the modification ofcell surfaces by altering glycosyl residues presented by those surfaces.For example, Fukuda and coworkers have developed a method for attachingglycosides of defined structure onto cell surfaces. The method exploitsthe relaxed substrate specificity of a fucosyltransferase that cantransfer fucose and fucose analogs bearing diverse glycosyl substrates(Tsuboi et al., J. Biol. Chem. 271: 27213 (1996)).

Enzymatic methods have also been used to activate glycosyl residues on aglycopeptide towards subsequent chemical elaboration. The glycosylresidues are typically activated using galactose oxidase, which convertsa terminal galactose residue to the corresponding aldehyde. The aldehydeis subsequently coupled to an amine-containing modifying group. Forexample, Casares et al. (Nature Biotech. 19: 142 (2001)) have attacheddoxorubicin to the oxidized galactose residues of a recombinantMHCII-peptide chimera.

Glycosyl residues have also been modified to contain ketone groups. Forexample, Mahal and co-workers (Science 276: 1125 (1997)) have preparedN-levulinoyl mannosamine (“ManLev”), which has a ketone functionality atthe position normally occupied by the acetyl group in the naturalsubstrate. Cells were treated with the ManLev, thereby incorporating aketone group onto the cell surface. See, also Saxon et al., Science 287:2007 (2000); Hang et al., J. Am. Chem. Soc. 123: 1242 (2001); Yarema etal., J. Biol. Chem. 273: 31168 (1998); and Charter et al., Glycobiology10: 1049 (2000).

The methods of modifying cell surfaces have not been applied in theabsence of a cell to modify a glycosylated or non-glycosylated peptide.Further, the methods of cell surface modification are not utilized forthe enzymatic incorporation preformed modified glycosyl donor moietyinto a peptide. Moreover, none of the cell surface modification methodsare practical for producing glycosyl-modified peptides on an industrialscale.

Despite the efforts directed toward the enzymatic elaboration ofsaccharide structures, there remains still a need for an industriallypractical method for the modification of glycosylated andnon-glycosylated peptides with modifying groups such as water-solublepolymers, therapeutic moieties, biomolecules and the like. Of particularinterest are methods in which the modified peptide has improvedproperties, which enhance its use as a therapeutic or diagnostic agent.The present invention fulfills these and other needs.

SUMMARY OF THE INVENTION

The invention includes a multitude of methods of remodeling a peptide tohave a specific glycan structure attached thereto. Although specificglycan structures are described herein, the invention should not beconstrued to be limited to any one particular structure. In addition,although specific peptides are described herein, the invention shouldnot be limited by the nature of the peptide described, but rather shouldencompass any and all suitable peptides and variations thereof.

The description which follows discloses the preferred embodiments of theinvention and provides a written description of the claims appendedhereto. The invention encompasses any and all variations of theseembodiments that are or become apparent following a reading of thepresent specification.

The invention includes a cell-free, in vitro method of remodeling anerythropoietin (EPO) peptide, the peptide having the formula:

wherein

-   -   AA is a terminal or internal amino acid residue of the peptide;    -   X¹—X² is a saccharide covalently linked to the AA, wherein    -   X¹ is a first glycosyl residue; and    -   X² is a second glycosyl residue covalently linked to X¹, wherein        X¹ and X² are selected from monosaccharyl and oligosaccharyl        residues;        the method comprising:    -   (a) removing X² or a saccharyl subunit thereof from the peptide,        thereby forming a truncated glycan; and    -   (b) contacting the truncated glycan with at least one        glycosyltransferase and at least one glycosyl donor under        conditions suitable to transfer the at least one glycosyl donor        to the truncated glycan, thereby remodeling the EPO peptide.

The method further comprises:

-   -   (c) removing X¹, thereby exposing the AA; and    -   (d) contacting the AA with at least one glycosyltransferase and        at least one glycosyl donor under conditions suitable to        transfer the at least one glycosyl donor to the AA, thereby        remodeling the EPO peptide.

The method additionally comprises:

-   -   (e) prior to step (b), removing a group added to the saccharide        during post-translational modification.

In one aspect, the group is a member selected from phosphate, sulfate,carboxylate and esters thereof.

In one aspect of the method, the peptide has the formula:

wherein

-   -   Z is a member selected from O, S, NH and a crosslinker.

In one embodiment, at least one of the glycosyl donors comprises amodifying group.

In this and in other embodiments, the modifying group is a memberselected from the group consisting of a polymer, a therapeutic moiety, adetectable label, a reactive linker group, a targeting moiety, and apeptide. Preferably, the modifying group is a water soluble polymer,preferably, poly(ethylene glycol), wherein preferably, the poly(ethyleneglycol) has a molecular weight distribution that is essentiallyhomodisperse.

There is also provided a cell-free in vitro method of remodeling an EPOpeptide, the peptide having the formula:

wherein

-   -   X³, X⁴, X⁵, X⁶, X⁷, and X¹⁷ are independently selected        monosaccharyl or oligosaccharyl residues; and    -   a, b, c, d, e and x are independently selected from the integers        0, 1 and 2, with the proviso that at least one member selected        from a, b, c, d, and e and x are 1 or 2; the method comprising:    -   (a) removing at least one of X³, X⁴, X⁵, X⁶, X⁷, or X¹⁷, a        saccharyl subunit thereof from the peptide, thereby forming a        truncated glycan; and    -   (b) contacting the truncated glycan with at least one        glycosyltransferase and at least one glycosyl donor under        conditions suitable to transfer the at least one glycosyl donor        to the truncated glycan, thereby remodeling the EPO peptide.

In one embodiment, the removing of step (a) produces a truncated glycanin which a, b, c, e and x are each 0.

In another embodiment, X³, X⁵, and X⁷, are selected from the groupconsisting of (mannose)_(z) and (mannose)_(z)-(X⁸)_(y)

wherein

-   -   X⁸ is a glycosyl moiety selected from mono- and        oligo-saccharides;    -   y is an integer selected from 0 and 1; and    -   z is an integer between 1 and 20, wherein    -   when z is 3 or greater, (mannose)_(z) is selected from linear        and branched structures.

In a further embodiment, X⁴ is selected from the group consisting ofGlcNAc and xylose.

In yet another embodiment, X³, X⁵, and X⁷ are (mannose)_(u), wherein

u is selected from the integers between 1 and 20, and when u is 3 orgreater, (mannose)_(u) is selected from linear and branched structures.

In one aspect, at least one of the glycosyl donors comprises a modifyinggroup.

There is further provided a cell-free in vitro method of remodeling anEPO peptide comprising a glycan having the formula:

wherein

-   -   r, s, and t are integers independently selected from 0 and 1,        the method comprising:    -   (a) contacting the peptide with at least one glycosyltransferase        and at least one glycosyl donor under conditions suitable to        transfer the at least one glycosyl donor to the glycan, thereby        remodeling the EPO peptide.

In one aspect, at least one of the glycosyl donors comprises a modifyinggroup.

In another aspect, the peptide has the formula:

wherein

-   -   X⁹ and X¹⁰ are independently selected monosaccharyl or        oligosaccharyl residues; and    -   m, n and f are integers selected from 0 and 1.

Further, the peptide has the formula:

wherein

-   -   X¹¹ and X¹² are independently selected glycosyl moieties; and    -   r and x are integers independently selected from 0 and 1.

In another aspect, X¹¹ and X¹² are (mannose)_(q), wherein

-   -   q is selected from the integers between 1 and 20, and when q is        three or greater, (mannose)_(q) is selected from linear and        branched structures.

In addition, the peptide has the formula:

wherein

-   -   X¹³, X¹⁴, and X¹⁵ are independently selected glycosyl residues;        and    -   g, h, i, j, k, and p are independently selected from the        integers 0 and 1, with the proviso that at least one of g, h, i,        j, k and p is 1.

The method also includes wherein

-   -   X¹⁴ and X¹⁵ are members independently selected from GlcNAc and        Sia; and i and k are independently selected from the integers 0        and 1, with the proviso that at least one of i and k is 1 and if        k is 1, g, h and j are 0.

In an additional aspect, the peptide has the formula:

wherein

-   -   X¹⁶ is a member selected from:

wherein

-   -   s and i are integers independently selected from 0 and 1.

In one embodiment, the removing utilizes a glycosidase.

Also included is a cell-free, in vitro method of remodeling an EPOpeptide having the formula:

wherein

-   -   AA is a terminal or internal amino acid residue of the peptide;    -   X¹ is a glycosyl residue covalently linked to the AA, selected        from monosaccharyl and oligosaccharyl residues; and    -   u is an integer selected from 0 and 1,        the method comprising:

contacting the peptide with at least one glycosyltransferase and atleast one glycosyl donor under conditions suitable to transfer the atleast one glycosyl donor to the truncated glycan, wherein the glycosyldonor comprises a modifying group, thereby remodeling the EPO peptide.

The invention further provides a covalent conjugate between an EPOpeptide and a modifying group that alters a property of the peptide,wherein the modifying group is covalently attached to the peptide at apreselected glycosyl or amino acid residue of the peptide via an intactglycosyl linking group.

In one aspect, the modifying group is a member selected from the groupconsisting of a polymer, a therapeutic moiety, a detectable label, areactive linker group, a targeting moiety, and a peptide. In anotheraspect, the modifying group and an intact glycosyl linking groupprecursor are linked as a covalently attached unit to the peptide viathe action of an enzyme, the enzyme converting the precursor to theintact glycosyl linking group, thereby forming the conjugate. In afurther aspect, the covalent conjugate comprises a first modifying groupcovalently linked to a first residue of the peptide via a first intactglycosyl linking group, and a second glycosyl linking group linked to asecond residue of the peptide via a second intact glycosyl linkinggroup. In one embodiment, the first residue and the second residue arestructurally identical. In another embodiment, the first residue and thesecond residue have different structures. In an additional embodiment,the first residue and the second residue are glycosyl residues. Further,the first residue and the second residue are amino acid residues. Also,the peptide may be remodeled prior to forming the conjugate. In anotheraspect, the remodeled peptide is remodeled to introduce an acceptormoiety for the intact glycosyl linking group. In yet a further aspect,the modifying group is a water-soluble polymer. In one aspect, theintact glycosyl linking unit is a member selected from the groupconsisting of a sialic acid residue, a Gal residue, a GlcNAc residue,and a GalNAc residue.

There is a also provided a method of forming a covalent conjugatebetween a polymer and a glycosylated or non-glycosylated peptide,wherein the polymer is conjugated to the peptide via an intact glycosyllinking group interposed between and covalently linked to both thepeptide and the polymer, the method comprising:

contacting the peptide with a mixture comprising a nucleotide sugarcovalently linked to the polymer and a glycosyltransferase for which thenucleotide sugar is a substrate under conditions sufficient to form theconjugate, wherein the peptide is EPO.

In one aspect, the polymer is a water-soluble polymer. In anotheraspect, the glycosyl linking group is covalently attached to a glycosylresidue covalently attached to the peptide. In yet a further aspect, theglycosyl linking group is covalently attached to an amino acid residueof the peptide. In an additional aspect, the polymer comprises a memberselected from the group consisting of a polyalkylene oxide and apolypeptide. An in another aspect, the polyalkylene oxide ispoly(ethylene glycol).

In yet a further aspect, the glycosyltransferase is selected from thegroup consisting of sialyltransferase, galactosyltransferase,glucosyltransferase, GalNAc transferase, and a GlcNAc transferase.Additionally, the glycosyltransferase is recombinantly produced and maybe either a recombinant prokaryotic or eukaryotic enzyme.

In one embodiment, the nucleotide sugar is selected from the groupconsisting of UDP-glycoside, CMP-glycoside, and GDP-glycoside. Further,the nucleotide sugar is selected from the group consisting ofUDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glucosamine,UDP-N-acetylgalactosamine, UDP-N-acetylglucosamine, GDP-mannose,GDP-fucose, CMP-sialic acid, CMP-NeuAc. In addition, the glycosylatedpeptide is partially deglycosylated prior to the contacting. Further,the intact glycosyl linking group is a sialic acid residue. In addition,the method is performed in a cell-free environment. Also, the covalentconjugate is isolated, preferably by membrane filtration.

The invention further provides a composition for forming a conjugatebetween a peptide and a modified sugar, the composition comprising: anadmixture of a modified sugar, a glycosyltransferase, and a peptideacceptor substrate, wherein the modified sugar has covalently attachedthereto a member selected from a polymer, a therapeutic moiety and abiomolecule, wherein the peptide is EPO.

In addition, there is provided an EPO peptide remodeled by the methodsof the invention and pharmaceutical compositions comprising such EPOpeptides.

Also provided is a cell-free, in vitro method of remodeling a peptidehaving the formula:

wherein

-   -   AA is a terminal or internal amino acid residue of the peptide,        the method comprising:

contacting the peptide with at least one glycosyltransferase and atleast one glycosyl donor under conditions suitable to transfer the atleast one glycosyl donor to the amino acid residue, wherein the glycosyldonor comprises a modifying group, thereby remodeling the peptide,wherein the peptide is EPO.

In addition, there is provided a method for forming a conjugate betweenan EPO peptide and a modifying group, wherein the modifying group iscovalently attached to the EPO peptide through an intact glycosyllinking group, the EPO peptide comprising a glycosyl residue having aformula which is a member selected from:

wherein

-   -   a, b, c, d, i, n, o, p, q, r, s, t, and u are members        independently selected from 0 and 1;    -   e, f, g, and h are members independently selected from the        integers between 0 and 4;    -   j, k, l, and m are members independently selected from the        integers between 0 and 20;    -   v, w, x, y, and z are 0; and    -   R is a modifying group, a mannose or an oligomannose;        the method comprising:    -   (a) contacting the EPO peptide with a glycosyltransferase and a        modified glycosyl donor, comprising a glycosyl moiety which is a        substrate for the glycosyltransferase covalently linked to the        modifying group, under conditions appropriate for the formation        of the intact glycosyl linking group.

The method further comprises:

-   -   (b) prior to step (a), contacting the EPO peptide with a        sialidase under conditions appropriate to remove sialic acid        from the EPO peptide.

The method additionally comprises:

-   -   (c) contacting the product of step (a) with a sialyltransferase        and a sialic acid donor under conditions appropriate to transfer        sialic acid to the product.

The method further comprises:

-   -   (d) prior to step (a), contacting the EPO peptide with a        galactosidase operating synthetically under conditions        appropriate to add a galactose to the EPO peptide.

In addition, the method comprises:

-   -   (e) prior to step (a), contacting the EPO peptide with a        galactosyl transferase and a galactose donor under conditions        appropriate to transfer the galactose to the EPO peptide.

The method also comprises:

-   -   (f) contacting the product from step (e) with ST3Gal3 and a        sialic acid donor under conditions appropriate to transfer        sialic acid to the product.

The method also comprises:

-   -   (g) contacting the product from step (a) with a moiety that        reacts with the modifying group, thereby forming a conjugate        between the intact glycosyl linking group and the moiety.

In addition, the method comprises:

-   -   (h) prior to step (a), contacting the EPO peptide with        N-acetylglucosamine transferase and a GlcNAc donor under        conditions appropriate to transfer GlcNAc to the EPO peptide.

In one asepct, the modifying group is a member selected from a polymer,a toxin, a radioisotope, a therapeutic moiety and a glycoconjugate.

In another aspect,

-   -   a, b, c, d, e, f, g, n, and q are 1;    -   h is a member selected from the integers between 1 and 3;    -   i, j, k, l, m, o, p, r, s, t, and u are members independently        selected from 0 and 1; and    -   v, w, x, y and z are 0.

In yet another aspect,

-   -   a, b, c, d, f, h, j, k, l, m, q, s, u, v, w, x, y, and z are 0;        and    -   e, g, i, r, and t are members independently selected from 0 and        1.

In an additional aspect,

-   -   a, b, c, d, e, f, g, h, i, j, k, l, m, n, o, p, q, r, s, t, and        u are members independently selected from 0 and 1; and    -   v, w, x, y, and z are 0.

In a further aspect,

-   -   a, b, c, d, e, f, g, n, and q are 1;    -   h is a member selected from the integers between 1 and 3;    -   i, j, k, l, m, o, p, r, s, t, and u are members independently        selected from 0 and 1; and    -   v, w, x, y and z are 0.

In another aspect,

-   -   a, b, c, d, f, h, j, k, l, m, o, p, s, u, v, w, x, y, and z are        0; and    -   e, g, i, n, q, r, and t are independently selected from 0 and 1.

Additionally,

-   -   a, b, c, d, f, h, j, k, l, m, n, o, p, s, u, v, w, x, y, and z        are 0; and    -   e, g, i, q, r, and t are members independently selected from 0        and 1.

Further

-   -   q is 1;    -   a, b, c, d, e, f, g, h, i, n, r, s, t, and u are members        independently selected from 0 and 1;

and

-   -   j, k, l, m, o, p, v, w, x, y, and z are 0.

There is also provided an EPO peptide conjugate formed by the methods ofthe invention.

Further provided is an EPO peptide comprising one or more glycans,having a glycoconjugate molecule covalently attached to the peptide. Inone aspect, the one or more glycans is a monoantennary glycan. Inanother aspect, the one or more glycans is a biantennary glycan. In afurther aspect, the one or more glycans is a triantennary glycan. In yetanother aspect, the one or more glycans is at least a triantennaryglycan. In a further aspect, the one or more glycans comprises at leasttwo glycans comprising a mixture of mono or multiantennary glycans. Inyet another aspect, the one or more glycans is selected from an N-linkedglycan and an O-linked glycan. In a further aspect, the one or moreglycans is at least two glycans selected from an N-linked and anO-linked glycan. Additionally, the peptide is expressed in a cellselected from the group consisting of a prokaryotic cell and aeukaryotic cell, and the eukaryotic cell is selected from the groupconsisting of a mammalian cell, an insect cell and a yeast cell.

Also provided in the invention is a glycoPEGylated EPO peptidecomprising an EPO peptide and at least one glycan and at least onepoly(ethylene glycol) molecule covalently attached to the glycan,wherein the poly(ethylene glycol) molecule is added to the EPO using aglycosyltransferase. In one aspect, the glycoPEGylated EPO peptidecomprises at least one mono-antennary glycan. In another aspect, all ofthe glycans are N-linked and are mono-antennary. In a further aspect,all of the glycans are N-linked and at least one of the glycans comprisethe poly(ethylene glycol). In an additional aspect, more than one of theglycans comprises the poly(ethylene glycol). In a further aspect, all ofthe glycans are N-linked and all of the glycans comprise thepoly(ethylene glycol). Additionally, the glycoPEGylated peptidecomprises at least three mono-antennary glycaris having thepoly(ethylene glycol) covalently attached thereto.

There is further provided a glycoPEGylated EPO peptide, wherein the EPOpeptide comprises three or more glycans. In one aspect, at least one ofthe glycans comprises the poly(ethylene glycol) covalently attachedthereto. In another aspect, more than one of the glycans comprises thepoly(ethylene glycol) covalently attached thereto. In a further aspect,all of the glycans comprise the poly(ethylene glycol) covalentlyattached thereto. Additionally, the poly(ethylene glycol) is linked toat least one sugar moiety selected from the group consisting of fucose(Fuc), N-acetylglucosamine (GlcNAc), galactose (Gal) and a sialic acid(SA). Additionally, the sialic acid is N-acetylneuraminic acid. Further,the EPO peptide does not comprise an O-linked glycan. In addition, theEPO peptide comprises at least one O-linked glycan. Further, theO-linked peptide comprises the poly(ethylene glycol) covanently attachedthereto. In addition, the EPO peptide is recombinantly expressed in acell. Further, the cell is selected from the group consisting of aninsect cell, a yeast cell and a mammalian cell. Preferably, the themammalian cell is a CHO cell. In one aspect, the poly(ethylene glycol)has a molecular weight selected from the group consisting of about 1kDa, 2 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa and 40 kDa. In another aspect,the EPO peptide is selected from the group consisting of a naturallyoccurring EPO peptide and a mutated EPO peptide. In another aspect,mutated EPO peptide comprises the amino acid sequence of SEQ ID NO:73having at least one mutation selected from the group consisting ofArg¹³⁹ to Ala¹³⁹, Arg¹⁴³ to Ala¹⁴³ and Lys¹⁵⁴ to Ala¹⁵⁴.

There is also provided a method of making a glycoPEGylated EPO peptide,the method comprising the steps of:

-   -   (a) contacting an EPO peptide with a mixture comprising a        nucleotide sugar covalently linked to poly(ethylene glycol) and        a glycosyltransferase under conditions sufficient to transfer        the poly(ethylene glycol) to the EPO peptide.

In one embodiment, the sugar of the nucleotide sugar is selected fromthe group consisting of fucose (Fuc), N-acetylglucosamine (GlcNAc),galactose (Gal) and a sialic acid (SA). In another embodiment, thesialic acid is N-acetylneuraminic acid (NAN). In a further embodiment,the poly(ethylene glycol) has a molecular weight selected from the groupconsisting of about 1 kDa, 2 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa and 40kDa. In an additional embodiment, the EPO peptide is recombinantlyexpressed in a cell, where the cell may be selected from the groupconsisting of an insect cell, a yeast cell and a mammalian cell. In oneaspect, the EPO peptide is selected from the group consisting of anaturally occurring EPO peptide and a mutated EPO peptide. In anotheraspect, the mature EPO has the sequence of SEQ ID NO:73. In a furtheraspect, the mutated EPO peptide comprises the amino acid sequence of SEQID NO: 73 having at least one mutation selected from the groupconsisting of Arg¹³⁹ to Ala¹³⁹, Arg¹⁴³ to Ala¹⁴³ and Lys¹⁵⁴ to Ala¹⁵⁴.

In another aspect of the method of the invention, before step (a): themethod comprises:

-   -   (b) contacting the EPO peptide with a mixture comprising a        nucleotide-N-acetylglucosamine (GlcNAc) molecule and an        N-acetylglucosamine transferase (GnT) for which the        nucleotide-GlcNAc is a substrate under conditions sufficient to        form a bond between the GlcNAc and the EPO, wherein the GnT is        selected from the group consisting of GnT I, GnT II, GnT III,        GnT IV, GnT V and GnT VI.

In one aspect, the mixture comprises one GnT selected from the groupconsisting of GnT I, GnT II, GnT IV, GnT V and GnT VI. In anotheraspect, the glycoPEGylated EPO peptide comprises at least onemono-antennary glycan. In a further aspect, the sugar of the nucleotidesugar is galactose and the glycosyltransferase is galactosyl transferaseI (GalT I).

In an additional aspect of the method, before step (a) but after step(b):

-   -   (c) contacting the EPO peptide with a mixture comprising a        nucleotide galactose (Gal) and galactosyl transferase I (GalT I)        under conditions sufficient to transfer galactose to the EPO        peptide.

In one embodiment, in step (a), the sugar of the nucleotide sugar issialic acid and the glycosyltransferase is a sialyltransferase. Inanother embodiment, the sialic acid is N-acetylneuraminic acid (NAN). Ina further embodiment, the sialyltransferase is selected from the groupconsisting of α(2,3)sialyltransferase, α(2,6)sialyltransferase and(2,8)sialyltransferase.

There is also provided a glycoPEGylated EPO, the EPO comprising thesequence of SEQ ID NO:73. In one aspect, the EPO comprises the sequenceof SEQ ID NO:73 and further comprising a mutation in the sequence.

There is further provided in the invention a method of making aglycoPEGylated EPO peptide, the method comprising the steps of:

-   -   (a) contacting an EPO peptide with a mixture comprising a        nucleotide sugar covalently linked to poly(ethylene glycol) and        a glycosyltransferase under conditions sufficient to transfer        the poly(ethylene glycol) to the EPO peptide, wherein the        glycosyltransferase is a fucosyltransferase.

In one aspect, the fucosyltransferase is selected from the groupconsisting of fucosyltransferase I, fucosyltransferase III,fucosyltransferase IV, fucosyltransferase V, fucosyltransferase VI andfucosyltransferase VII, and in another, EPO is expressed in a CHO cell.

There is further provided a method of treating a mammal having anemia,the method comprising administering to the mammal an EPO peptide havingone or more glycans having a glycoconjugate molecule attached to thepeptide, wherein the EPO is administered in an amount effective toincrease the hematocrit level in the mammal. In this and in otherembodiments, the mammal is a human.

Also provided is a method of providing erythropoietin therapy to amammal, the method comprising administering an effective amount of aglycoPEGylated EPO peptide comprising an EPO peptide and at least oneglycan and at least one poly(ethylene glycol) molecule covalentlyattached to the glycan, wherein the poly(ethylene glycol) molecule isadded to the EPO using a glycosyltransferase, wherein the EPO isadministered in an amount effective to increase the hematocrit level inthe mammal.

In addition, there is provided a method of treating a mammal havinganemia, the method comprising administering to the mammal aglycoPEGylated EPO peptide comprising an EPO peptide and at least oneglycan and at least one poly(ethylene glycol) molecule covalentlyattached to the glycan, wherein the poly(ethylene glycol) molecule isadded to the EPO using a glycosyltransferase, wherein the EPO isadministered in an amount effective to increase the hematocrit level inthe mammal. In one aspect, the anemia is associated with chemotherapy.

Further provided is a method of treating a kidney dialysis patient, themethod comprising administering to the patient a glycoPEGylated EPOpeptide comprising an EPO peptide and at least one glycan and at leastone poly(ethylene glycol) molecule covalently attached to the glycan,wherein the poly(ethylene glycol) molecule is added to the EPO using aglycosyltransferase, wherein the EPO is administered in an amounteffective to increase the hematocrit level in the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 is a scheme depicting a trimannosyl core glycan (left side) andthe enzymatic process for the generation of a glycan having a bisectingGlcNAc (right side).

FIG. 2 is a scheme depicting an elemental trimannosyl core structure andcomplex chains in various degrees of completion. The in vitro enzymaticgeneration of an elemental trimannosyl core structure from a complexcarbohydrate glycan structure which does not contain a bisecting GlcNAcresidue is shown, as is the generation of a glycan structure therefromwhich contains a bisecting GlcNAc. Symbols: squares: GlcNAc; lightcircles: Man; dark circles: Gal; triangles: NeuAc.

FIG. 3 is a scheme for the enzymatic generation of a sialylated glycanstructure (right side) beginning with a glycan having a trimannosyl coreand a bisecting GlcNAc (left side).

FIG. 4 is a scheme of a typical high mannose containing glycan structure(left side) and the enzymatic process for reduction of this structure toan elemental trimannosyl core structure. In this scheme, X is mannose asa monosaccharide, an oligosaccharide or a polysaccharide.

FIG. 5 is a diagram of a fucose and xylose containing N-linked glycanstructure typically produced in plant cells.

FIG. 6 is a diagram of a fucose containing N-linked glycan structuretypically produced in insect cells. Note that the glycan may have nocore fucose, it amy have a single core fucose with either linkage, or itmay have a single core fucose having a preponderance of one linkage.

FIG. 7 is a scheme depicting a variety of pathways for the trimming of ahigh mannose structure and the synthesis of complex sugar chainstherefrom. Symbols: squares: GlcNAc; circles: Man; diamonds: fucose;pentagon: xylose.

FIG. 8 is a scheme depicting in vitro strategies for the synthesis ofcomplex structures from an elemental trimannosyl core structure.Symbols: Squares: GlcNAc; light circles: Man; dark circles: Gal; darktriangles: NeuAc; GnT: N-acetyl glucosaminyltransferase; GalT:galactosyltransferase; ST: sialyltransferase.

FIG. 9 is a scheme depicting two in vitro strategies for the synthesisof monoantennary glycans, and the optional glycoPEGylation of the same.Dark squares: GlcNAc; dark circles: Man; light circles: Gal; darktriangles: sialic acid.

FIG. 10 is a scheme depicting two in vitro strategies for the synthesisof monoantennary glycans, and the optional glycoPEGylation of the same.Dark squares: GlcNAc; dark circles: Man; light circles: Gal; darktriangles: sialic acid.

FIG. 11 is a scheme depicting various complex structures, which may besynthesized from an elemental trimannosyl core structure. Symbols:Squares: GlcNAc; light circles: Man; dark circles: Gal; triangles:NeuAc; diamonds: fucose; FT and FucT: fucosyltransferase; GalT:galactosyltransferase; ST: sialyltransferase; Le: Lewis antigen; SLe:sialylated Lewis antigen.

FIG. 12 is an exemplary scheme for preparing O-linked glycopeptidesoriginating with serine or threonine. Optionally, a water solublepolymer (WSP) such as poly(ethylene glycol) is added to the final glycanstructure.

FIG. 13 is a series of diagrams depicting the four types of O-glycanstructures, termed cores 1 through 4. The core structure is outlined indotted lines.

FIG. 14, comprising FIG. 14A and FIG. 14B, is a series of schemesshowing an exemplary embodiment of the invention in which carbohydrateresidues comprising complex carbohydrate structures and/or high mannosehigh mannose structures are trimmed back to the first generationbiantennary structure. Optionally, fucose is added only after reactionwith GnT I. A modified sugar bearing a water-soluble polymer (WSP) isthen conjugated to one or more of the sugar residues exposed by thetrimming back process.

FIG. 15 is a scheme similar to that shown in FIG. 4, in which a highmannose or complex structure is “trimmed back” to the mannosebeta-linked core and a modified sugar bearing a water soluble polymer isthen conjugated to one or more of the sugar residues exposed by thetrimming back process. Sugars are added sequentially usingglycosyltransferases.

FIG. 16 is a scheme similar to that shown in FIG. 4, in which a highmannose or complex structure is trimmed back to the GlcNAc to which thefirst mannose is attached, and a modified sugar bearing a water solublepolymer is then conjugated to one or more of the sugar residues exposedby the trimming back process. Sugars are added sequentially usingglycosyltransferases.

FIG. 17 is a scheme similar to that shown in FIG. 4, in which a highmannose or cpomplex structure is trimmed back to the first GlcNAcattached to the Asn of the peptide, following which a water solublepolymer is conjugated to one or more sugar residues which havesubsequently been added on. Sugars are added sequentially usingglycosyltransferases.

FIG. 18, comprising FIGS. 18A and 18B, is a scheme in which an N-linkedcarbohydrate is optionally trimmed back from a high mannose or cpmplexstructure, and subsequently derivatized with a modified sugar moiety(Gal or GlcNAc) bearing a water-soluble polymer.

FIG. 19, comprising FIGS. 19A and 19B, is a scheme in which an N-linkedcarbohydrate is trimmed back from a high mannose or complex structureand subsequently derivatized with a sialic acid moiety bearing awater-soluble polymer. Sugars are added sequentially usingglycosyltransferases.

FIG. 20 is a scheme in which an N-linked carbohydrate is optionallytrimmed back from a high mannose or complex structure and subsequentlyderivatized with one or more sialic acid moieties, and terminated with asialic acid derivatized with a water-soluble polymer. Sugars are addedsequentially using glycosyltransferases.

FIG. 21 is a scheme in which an O-linked saccharide is “trimmed back”and subsequently conjugated to a modified sugar bearing a water-solublepolymer. In the exemplary scheme, the carbohydrate moiety is “trimmedback” to the first generation of the biantennary structure.

FIG. 22 is an exemplary scheme for trimming back the carbohydrate moietyof an O-linked glycopeptide to produce a mannose available forconjugation with a modified sugar having a water-soluble polymerattached thereto.

FIG. 23, comprising FIG. 23A to FIG. 23C, is a series of exemplaryschemes. FIG. 23A is a scheme that illustrates addition of a PEGylatedsugar, followed by the addition of a non-modified sugar. FIG. 23B is ascheme that illustrates the addition of more that one kind of modifiedsugar onto one glycan. FIG. 23C is a scheme that illustrates theaddition of different modified sugars onto O-linked glycans and N-linkedglycans.

FIG. 24 is a diagram of various methods of improving the therapeuticfunction of a peptide by glycan remodeling, including conjugation.

FIG. 25 is a set of schemes for glycan remodeling of a therapeuticpeptide to treat Gaucher Disease.

FIG. 26 is a scheme for glycan remodeling to generate glycans having aterminal mannose-6-phosphate moiety.

FIG. 27 is a diagram illustrating the array of glycan structures foundon CHO-produced glucocerebrosidase (Cerezyme™) after sialylation.

FIG. 28, comprising FIG. 28A to FIG. 28Z and FIG. 28AA to FIG. 28CC, isa list of peptides useful in the methods of the invention.

FIG. 29, comprising FIGS. 29A to 29G, provides exemplary schemes forremodeling glycan structures on granulocyte colony stimulating factor(G-CSF). FIG. 29A is a diagram depicting the G-CSF peptide indicatingthe amino acid residue to which a glycan is bonded, and an exemplaryglycan formula linked thereto. FIG. 29B to 29G are diagrams ofcontemplated remodeling steps of the glycan of the peptide in FIG. 29Abased on the type of cell the peptide is expressed in and the desiredremodeled glycan structure.

FIG. 30, comprising FIGS. 30A to 30EE sets forth exemplary schemes forremodeling glycan structures on interferon-alpha. FIG. 30A is a diagramdepicting the interferon-alpha isoform 14c peptide indicating the aminoacid residue to which a glycan is bonded, and an exemplary glycanformula linked thereto. FIG. 30B to 30D are diagrams of contemplatedremodeling steps of the glycan of the peptide in FIG. 30A based on thetype of cell the peptide is expressed in and the desired remodeledglycan structure. FIG. 30E is a diagram depicting the interferon-alphaisoform 14c peptide indicating the amino acid residue to which a glycanis linked, and an exemplary glycan formula linked thereto. FIG. 30F to30N are diagrams of contemplated remodeling steps of the glycan of thepeptide in FIG. 30E based on the type of cell the peptide is expressedin and the desired remodeled glycan structure. FIG. 30O is a diagramdepicting the interferon-alpha isoform 2a or 2b peptides indicating theamino acid residue to which a glycan is linked, and an exemplary glycanformula linked thereto. FIG. 30P to 30W are diagrams of contemplatedremodeling steps of the glycan of the peptide in FIG. 30O based on thetype of cell the peptide is expressed in and the desired remodeledglycan structure. FIG. 30X is a diagram depicting theinterferon-alpha-mucin fusion peptides indicating the residue(s) whichis linked to glycans contemplated for remodeling, and exemplary glycanformulas linked thereto. FIG. 30Y to 30AA are diagrams of contemplatedremodeling steps of the glycan of the peptides in FIG. 30X based on thetype of cell the peptide is expressed in and the desired remodeledglycan structure. FIG. 30BB is a diagram depicting theinterferon-alpha-mucin fusion peptides and interferon-alpha peptidesindicating the residue(s) which bind to glycans contemplated forremodeling, and formulas for the glycans. FIG. 30CC to 30EE are diagramsof contemplated remodeling steps of the glycan of the peptides in FIG.30BB based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure.

FIG. 31, comprising FIGS. 31A to 31S, sets forth exemplary schemes forremodeling glycan structures on interferon-beta. FIG. 31A is a diagramdepicting the interferon-beta peptide indicating the amino acid residueto which a glycan is linked, and an exemplary glycan formula linkedthereto. FIG. 31B to 31O are diagrams of contemplated remodeling stepsof the glycan of the peptide in FIG. 31A based on the type of cell thepeptide is expressed in and the desired remodeled glycan structure. FIG.31P is a diagram depicting the interferon-beta peptide indicating theamino acid residue to which a glycan is linked, and an exemplary glycanformula linked thereto. FIG. 31Q to 31S are diagrams of contemplatedremodeling steps of the glycan of the peptide in FIG. 31P based on thetype of cell the peptide is expressed in and the desired remodeledglycan structure.

FIG. 32, comprising FIGS. 32A to 32D, sets forth exemplary schemes forremodeling glycan structures on Factor VII and Factor VIIa. FIG. 32A isa diagram depicting the Factor-VII and Factor-VIIa peptides A (solidline) and B (dotted line) indicating the residues which bind to glycanscontemplated for remodeling, and the formulas for the glycans. FIG. 32Bto 32D are diagrams of contemplated remodeling steps of the glycan ofthe peptide in FIG. 32A based on the type of cell the peptide isexpressed in and the desired remodeled glycan structure.

FIG. 33, comprising FIGS. 33A to 33G, sets forth exemplary schemes forremodeling glycan structures on Factor IX. FIG. 33A is a diagramdepicting the Factor-IX peptide indicating residues which bind toglycans contemplated for remodeling, and formulas of the glycans. FIG.33B to 33G are diagrams of contemplated remodeling steps of the glycanof the peptide in FIG. 33A based on the type of cell the peptide isexpressed in and the desired remodeled glycan structure.

FIG. 34, comprising FIGS. 34A to 34J, sets forth exemplary schemes forremodeling glycan structures on follicle stimulating hormone (FSH),comprising α and β subunits. FIG. 34A is a diagram depicting theFollicle Stimulating Hormone peptides FSHα and FSHβ indicating theresidues which bind to glycans contemplated for remodeling, andexemplary glycan formulas linked thereto. FIG. 34B to 34J are diagramsof contemplated remodeling steps of the glycan of the peptides in FIG.34A based on the type of cell the peptides are expressed in and thedesired remodeled glycan structures.

FIG. 35, comprising FIGS. 35A to 35AA, sets forth exemplary schemes forremodeling glycan structures on Erythropoietin (EPO). FIG. 35A is adiagram depicting the EPO peptide indicating the residues which bind toglycans contemplated for remodeling, and formulas for the glycans. FIG.35B to 35S are diagrams of contemplated remodeling steps of the glycanof the peptide in FIG. 35A based on the type of cell the peptide isexpressed in and the desired remodeled glycan structure. FIG. 35T is adiagram depicting the EPO peptide indicating the residues which bind toglycans contemplated for remodeling, and formulas for the glycans. FIG.35U to 35W are diagrams of contemplated remodeling steps of the glycanof the peptide in FIG. 35T based on the type of cell the peptide isexpressed in and the desired remodeled glycan structure. FIG. 35X is adiagram depicting the EPO peptide indicating the residues which bind toglycans contemplated for remodeling, and formulas for the glycans. FIG.35Y to 35AA are diagrams of contemplated remodeling steps of the glycanof the peptide in FIG. 35X based on the type of cell the peptide isexpressed in and the desired remodeled glycan structure.

FIG. 36, comprising FIGS. 36A to 36K sets forth exemplary schemes forremodeling glycan structures on Granulocyte-Macrophage ColonyStimulating Factor (GM-CSF). FIG. 36A is a diagram depicting the GM-CSFpeptide indicating the residues which bind to glycans contemplated forremodeling, and formulas for the glycans. FIG. 36B to 36G are diagramsof contemplated remodeling steps of the glycan of the peptide in FIG.36A based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure. FIG. 36H is a diagram depicting theGM-CSF peptide indicating the residues which bind to glycanscontemplated for remodeling, and formulas for the glycans. FIG. 36I to36K are diagrams of contemplated remodeling steps of the glycan of thepeptide in FIG. 36H based on the type of cell the peptide is expressedin and the desired remodeled glycan structure.

FIG. 37, comprising FIGS. 37A to 37N, sets forth exemplary schemes forremodeling glycan structures on interferon-gamma. FIG. 37A is a diagramdepicting an interferon-gamma peptide indicating the residues which bindto glycans contemplated for remodeling, and exemplary glycan formulaslinked thereto. FIG. 37B to 37G are diagrams of contemplated remodelingsteps of the peptide in FIG. 37A based on the type of cell the peptideis expressed in and the desired remodeled glycan structure. FIG. 37H isa diagram depicting an interferon-gamma peptide indicating the residueswhich bind to glycans contemplated for remodeling, and exemplary glycanformulas linked thereto. FIG. 37I to 37N are diagrams of contemplatedremodeling steps of the peptide in FIG. 37H based on the type of cellthe peptide is expressed in and the desired remodeled glycan structure.

FIG. 38, comprising FIGS. 38A to 38N, sets forth exemplary schemes forremodeling glycan structures on α₁-antitrypsin (AIT, or α-1 proteaseinhibitor). FIG. 38A is a diagram depicting an AAT peptide indicatingthe residues which bind to glycans contemplated for remodeling, andexemplary glycan formulas linked thereto. FIG. 38B to 38F are diagramsof contemplated remodeling steps of the glycan of the peptide in FIG.38A based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure. FIG. 38G is a diagram depicting anAAT peptide indicating the residues which bind to glycans contemplatedfor remodeling, and exemplary glycan formulas linked thereto. FIG. 38Hto 38J are diagrams of contemplated remodeling steps of the peptide inFIG. 38G based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure. FIG. 38K is a diagram depicting anAAT peptide indicating the residues which bind to glycans contemplatedfor remodeling, and exemplary glycan formulas linked thereto. FIG. 38Lto 38N are diagrams of contemplated remodeling steps of the peptide inFIG. 38K based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure.

FIG. 39, comprising FIGS. 39A to 39J sets forth exemplary schemes forremodeling glycan structures on glucocerebrosidase. FIG. 39A is adiagram depicting the glucocerebrosidase peptide indicating the residueswhich bind to glycans contemplated for remodeling, and exemplary glycanformulas linked thereto. FIG. 39B to 39F are diagrams of contemplatedremodeling steps of the glycan of the peptide in FIG. 39A based on thetype of cell the peptide is expressed in and the desired remodeledglycan structure. FIG. 39G is a diagram depicting the glucocerebrosidasepeptide indicating the residues which bind to glycans contemplated forremodeling, and exemplary glycan formulas linked thereto. FIG. 39H to39K are diagrams of contemplated remodeling steps of the glycan of thepeptide in FIG. 39G based on the type of cell the peptide is expressedin and the desired remodeled glycan structure.

FIG. 40, comprising FIGS. 40A to 40W, sets forth exemplary schemes forremodeling glycan structures on Tissue-Type Plasminogen Activator (TPA).FIG. 40A is a diagram depicting the TPA peptide indicating the residueswhich bind to glycans contemplated for remodeling, and formulas for theglycans. FIG. 40B to 40G are diagrams of contemplated remodeling stepsof the peptide in FIG. 40A based on the type of cell the peptide isexpressed in and the desired remodeled glycan structure. FIG. 40H is adiagram depicting the TPA peptide indicating the residues which bind toglycans contemplated for remodeling, and formulas for the glycans. FIG.40I to 40K are diagrams of contemplated remodeling steps of the peptidein FIG. 40H based on the type of cell the peptide is expressed in andthe desired remodeled glycan structure. FIG. 40L is a diagram depictinga mutant TPA peptide indicating the residues which bind to glycanscontemplated for remodeling, and the formula for the glycans. FIG. 40Mto 40O are diagrams of contemplated remodeling steps of the peptide inFIG. 40L based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure. FIG. 40P is a diagram depicting amutant TPA peptide indicating the residues which bind to glycanscontemplated for remodeling, and formulas for the glycans. FIG. 40Q to40S are diagrams of contemplated remodeling steps of the peptide in FIG.40P based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure. FIG. 40T is a diagram depicting amutant TPA peptide indicating the residues which links to glycanscontemplated for remodeling, and formulas for the glycans. FIG. 40U to40W are diagrams of contemplated remodeling steps of the peptide in FIG.40T based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure.

FIG. 41, comprising FIGS. 41A to 41G, sets forth exemplary schemes forremodeling glycan structures on Interleukin-2 (IL-2). FIG. 41A is adiagram depicting the Interleukin-2 peptide indicating the amino acidresidue to which a glycan is linked, and an exemplary glycan formulalinked thereto. FIG. 41B to 41G are diagrams of contemplated remodelingsteps of the glycan of the peptide in FIG. 41A based on the type of cellthe peptide is expressed in and the desired remodeled glycan structure.

FIG. 42, comprising FIGS. 42A to 42M, sets forth exemplary schemes forremodeling glycan structures on Factor VIII. FIG. 42A are the formulasfor the glycans that bind to the N-linked glycosylation sites (A and A′)and to the O-linked sites (B) of the Factor VIII peptides. FIG. 42B to42F are diagrams of contemplated remodeling steps of the peptides inFIG. 42A based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure. FIG. 42G are the formulas for theglycans that bind to the N-linked glycosylation sites (A and A′) and tothe O-linked sites (B) of the Factor VIII peptides. FIG. 42H to 42M arediagrams of contemplated remodeling steps of the peptides in FIG. 42Gbased on the type of cell the peptide is expressed in and the desiredremodeled glycan structures.

FIG. 43, comprising FIGS. 43A to 43L, sets forth exemplary schemes forremodeling glycan structures on urokinase. FIG. 43A is a diagramdepicting the urokinase peptide indicating a residue which is linked toa glycan contemplated for remodeling, and an exemplary glycan formulalinked thereto. FIG. 43B to 43F are diagrams of contemplated remodelingsteps of the peptide in FIG. 43A based on the type of cell the peptideis expressed in and the desired remodeled glycan structure. FIG. 43G isa diagram depicting the urokinase peptide indicating a residue which islinked to a glycan contemplated for remodeling, and an exemplary glycanformula linked thereto. FIG. 43H to 43L are diagrams of contemplatedremodeling steps of the peptide in FIG. 43G based on the type of cellthe peptide is expressed in and the desired remodeled glycan structure.

FIG. 44, comprising FIGS. 44A to 44J, sets forth exemplary schemes forremodeling glycan structures on human DNase (hDNase). FIG. 44A is adiagram depicting the human DNase peptide indicating the residues whichbind to glycans contemplated for remodeling, and exemplary glycanformulas linked thereto. FIG. 44B to 44F are diagrams of contemplatedremodeling steps of the peptide in FIG. 44A based on the type of cellthe peptide is expressed in and the desired remodeled glycan structure.FIG. 44G is a diagram depicting the human DNase peptide indicatingresidues which bind to glycans contemplated for remodeling, andexemplary glycan formulas linked thereto. FIG. 44H to 44J are diagramsof contemplated remodeling steps of the peptide in FIG. 44F based on thetype of cell the peptide is expressed in and the desired remodeledglycan structure.

FIG. 45, comprising FIGS. 45A to 45L, sets forth exemplary schemes forremodeling glycan structures on insulin. FIG. 45A is a diagram depictingthe insulin peptide mutated to contain an N glycosylation site and anexemplary glycan formula linked thereto. FIG. 45B to 45D are diagrams ofcontemplated remodeling steps of the peptide in FIG. 45A based on thetype of cell the peptide is expressed in and the desired remodeledglycan structure. FIG. 45E is a diagram depicting insulin-mucin fusionpeptides indicating a residue(s) which is linked to a glycancontemplated for remodeling, and an exemplary glycan formula linkedthereto. FIG. 45F to 45H are diagrams of contemplated remodeling stepsof the peptide in FIG. 45E based on the type of cell the peptide isexpressed in and the desired remodeled glycan structure. FIG. 451 is adiagram depicting the insulin-mucin fusion peptides and insulin peptidesindicating a residue(s) which is linked to a glycan contemplated forremodeling, and formulas for the glycan. FIG. 45J to 45L are diagrams ofcontemplated remodeling steps of the peptide in FIG. 45I based on thetype of cell the peptide is expressed in and the desired remodeledglycan structure.

FIG. 46, comprising FIGS. 46A to 46K, sets forth exemplary schemes forremodeling glycan structures on the M-antigen (preS and S) of theHepatitis B surface protein (HbsAg). FIG. 46A is a diagram depicting theM-antigen peptide indicating the residues which bind to glycanscontemplated for remodeling, and formulas for the glycans. FIG. 46B to46G are diagrams of contemplated remodeling steps of the peptide in FIG.46A based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure. FIG. 46H is a diagram depicting theM-antigen peptide indicating the residues which bind to glycanscontemplated for remodeling, and formulas for the glycans. FIG. 46I to46K are diagrams of contemplated remodeling steps of the peptide in FIG.46H based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure.

FIG. 47, comprising FIGS. 47A to 47K, sets forth exemplary schemes forremodeling glycan structures on human growth hormone, including N, V andvariants thereof. FIG. 47A is a diagram depicting the human growthhormone peptide indicating a residue which is linked to a glycancontemplated for remodeling, and an exemplary glycan formula linkedthereto. FIG. 47B to 47D are diagrams of contemplated remodeling stepsof the glycan of the peptide in FIG. 47A based on the type of cell thepeptide is expressed in and the desired remodeled glycan structure. FIG.47E is a diagram depicting the three fusion peptides comprising thehuman growth hormone peptide and part or all of a mucin peptide, andindicating a residue(s) which is linked to a glycan contemplated forremodeling, and exemplary glycan formula(s) linked thereto. FIG. 47F to47K are diagrams of contemplated remodeling steps of the glycan of thepeptides in FIG. 47E based on the type of cell the peptide is expressedin and the desired remodeled glycan structure.

FIG. 48, comprising FIGS. 48A to 48G, sets forth exemplary schemes forremodeling glycan structures on a TNF Receptor-IgG Fc region fusionprotein (Enbrel™). FIG. 48A is a diagram depicting a TNF Receptor—IgG Fcregion fusion peptide which may be mutated to contain additionalN-glycosylation sites indicating the residues which bind to glycanscontemplated for remodeling, and formulas for the glycans. The TNFreceptor peptide is depicted in bold line, and the IgG Fc regions isdepicted in regular line. FIG. 48B to 48G are diagrams of contemplatedremodeling steps of the peptide in FIG. 48A based on the type of cellthe peptide is expressed in and the desired remodeled glycan structure.

FIG. 49, comprising FIGS. 49A to 49D, sets forth exemplary schemes forremodeling glycan structures on an anti-HER2 monoclonal antibody(Herceptin™). FIG. 49A is a diagram depicting an anti-HER2 monoclonalantibody which has been mutated to contain an N-glycosylation site(s)indicating a residue(s) on the antibody heavy chain which is linked to aglycan contemplated for remodeling, and an exemplary glycan formulalinked thereto. FIG. 49B to 49D are diagrams of contemplated remodelingsteps of the glycan of the peptides in FIG. 49A based on the type ofcell the peptide is expressed in and the desired remodeled glycanstructure.

FIG. 50, comprising FIGS. 50A to 50D, sets forth exemplary schemes forremodeling glycan structures on a monoclonal antibody to Protein F ofRespiratory Syncytial Virus (Synagis™). FIG. 50A is a diagram depictinga monoclonal antibody to Protein F peptide which is mutated to containan N-glycosylation site(s) indicating a residue(s) which is linked to aglycan contemplated for remodeling, and an exemplary glycan formulalinked thereto. FIG. 50B to 50D are diagrams of contemplated remodelingsteps of the peptide in FIG. 50A based on the type of cell the peptideis expressed in and the desired remodeled glycan structure.

FIG. 51, comprising FIGS. 51A to 51D, sets forth exemplary schemes forremodeling glycan structures on a monoclonal antibody to TNF-α(Remicade™). FIG. 51A is a diagram depicting a monoclonal antibody toTNF-α which has an N-glycosylation site(s) indicating a residue which islinked to a glycan contemplated for remodeling, and an exemplary glycanformula linked thereto. FIG. 51B to 51D are diagrams of contemplatedremodeling steps of the peptide in FIG. 51A based on the type of cellthe peptide is expressed in and the desired remodeled glycan structure.

FIG. 52, comprising FIGS. 52A to 52L, sets forth exemplary schemes forremodeling glycan structures on a monoclonal antibody to glycoproteinIIb/IIIa (Reopro™). FIG. 52A is a diagram depicting a mutant monoclonalantibody to glycoprotein IIb/IIIa peptides which have been mutated tocontain an N-glycosylation site(s) indicating the residue(s) which bindto glycans contemplated for remodeling, and exemplary glycan formulaslinked thereto. FIG. 52B to 52D are diagrams of contemplated remodelingsteps based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure. FIG. 52E is a diagram depictingmonoclonal antibody to glycoprotein IIb/IIIa-mucin fusion peptidesindicating the residues which bind to glycans contemplated forremodeling, and exemplary glycan formulas linked thereto. FIG. 52F to52H are diagrams of contemplated remodeling steps based on the type ofcell the peptide is expressed in and the desired remodeled glycanstructure. FIG. 52I is a diagram depicting monoclonal antibody toglycoprotein IIb/IIIa-mucin fusion peptides and monoclonal antibody toglycoprotein IIb/IIIa peptides indicating the residues which bind toglycans contemplated for remodeling, and exemplary glycan formulaslinked thereto. FIG. 52J to 52L are diagrams of contemplated remodelingsteps based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure.

FIG. 53, comprising FIGS. 53A to 53G, sets forth exemplary schemes forremodeling glycan structures on a monoclonal antibody to CD20(Rituxan™). FIG. 53A is a diagram depicting monoclonal antibody to CD20which have been mutated to contain an N-glycosylation site(s) indicatingthe residue which is linked to glycans contemplated for remodeling, andexemplary glycan formulas linked thereto. FIG. 53B to 53D are diagramsof contemplated remodeling steps of the glycan of the peptides in FIG.53A based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure. FIG. 53E is a diagram depictingmonoclonal antibody to CD20 which has been mutated to contain anN-glycosylation site(s) indicating the residue(s) which is linked toglycans contemplated for remodeling, and exemplary glycan formulaslinked thereto. FIG. 53F to 53G are diagrams of contemplated remodelingsteps of the glycan of the peptides in FIG. 53E based on the type ofcell the peptide is expressed in and the desired remodeled glycanstructure.

FIG. 54, comprising FIGS. 54A to 54O, sets forth exemplary schemes forremodeling glycan structures on anti-thrombin III (AT III). FIG. 54A isa diagram depicting the anti-thrombin III peptide indicating the aminoacid residues to which an N-linked glycan is linked, and an exemplaryglycan formula linked thereto. FIG. 54B to 54G are diagrams ofcontemplated remodeling steps of the glycan of the peptide in FIG. 54Abased on the type of cell the peptide is expressed in and the desiredremodeled glycan structure. FIG. 54H is a diagram depicting theanti-thrombin III peptide indicating the amino acid residues to which anN-linked glycan is linked, and an exemplary glycan formula linkedthereto. FIG. 54I to 54K are diagrams of contemplated remodeling stepsof the glycan of the peptide in FIG. 54H based on the type of cell thepeptide is expressed in and the desired remodeled glycan structure. FIG.54L is a diagram depicting the anti-thrombin III peptide indicating theamino acid residues to which an N-linked glycan is linked, and anexemplary glycan formula linked thereto. FIG. 54M to 54O are diagrams ofcontemplated remodeling steps of the glycan of the peptide in FIG. 54Lbased on the type of cell the peptide is expressed in and the desiredremodeled glycan structure.

FIG. 55, comprising FIGS. 55A to 55J, sets forth exemplary schemes forremodeling glycan structures on subunits α and β of human ChorionicGonadotropin (hCG).

FIG. 55A is a diagram depicting the hCGa and hCGP peptides indicatingthe residues which bind to N-linked glycans (A) and O-linked glycans (B)contemplated for remodeling, and formulas for the glyeans. FIG. 55B to55J are diagrams of contemplated remodeling steps based on the type ofcell the peptide is expressed in and the desired remodeled glycanstructure.

FIG. 56, comprising FIGS. 56A to 56J, sets forth exemplary schemes forremodeling glycan structures on alpha-galactosidase (Fabrazyme™). FIG.56A is a diagram depicting the alpha-galactosidase A peptide indicatingthe amino acid residues which bind to N-linked glycans (A) contemplatedfor remodeling, and formulas for the glycans. FIG. 56B to 56J arediagrams of contemplated remodeling steps based on the type of cell, thepeptide is expressed in and the desired remodeled glycan structure.

FIG. 57, comprising FIGS. 57A to 57J, sets forth exemplary schemes forremodeling glycan structures on alpha-iduronidase (Aldurazyme™). FIG.57A is a diagram depicting the alpha-iduronidase peptide indicating theamino acid residues which bind to N-linked glycans (A) contemplated forremodeling, and formulas for the glycans. FIG. 57B to 57J are diagramsof contemplated remodeling steps based on the type of cell the peptideis expressed in and the desired remodeled glycan structure.

FIG. 58, comprising FIGS. 58A and 58B, is an exemplary nucleotide andcorresponding amino acid sequence of granulocyte colony stimulatingfactor (G-CSF) (SEQ ID NOS: 1 and 2, respectively).

FIG. 59, comprising FIGS. 59A through 59D, is an exemplary nucleotideand corresponding amino acid sequence of interferon alpha (IFN-alpha)(FIGS. 59A and 59B, SEQ ID NOS: 3 and 4, respectively), and an exemplarynucleotide and corresponding amino acid sequence of interferon-omega(IFN-omega) (FIGS. 59C and 59D, SEQ ID NOS: 74 and 75, respectively).

FIG. 60, comprising FIGS. 60A and 60B, is an exemplary nucleotide andcorresponding amino acid sequence of interferon beta (IFN-beta) (SEQ IDNOS: 5 and 6, respectively).

FIG. 61, comprising FIGS. 61A and 61B, is an exemplary nucleotide andcorresponding amino acid sequence of Factor VIIa (SEQ ID NOS: 7 and 8,respectively).

FIG. 62, comprising FIGS. 62A and 62B, is an exemplary nucleotide andcorresponding amino acid sequence of Factor IX (SEQ ID NOS: 9 and 10,respectively).

FIG. 63, comprising FIGS. 63A through 63D, is an exemplary nucleotideand corresponding amino acid sequence of the alpha and beta chains offollicle stimulating hormone (FSH), respectively (SEQ ID NOS: 11 through14, respectively).

FIG. 64, comprising FIGS. 64A and 64B, is an exemplary nucleotide andcorresponding amino acid sequence of erythropoietin (EPO) (SEQ ID NOS:15 and 16, respectively).

FIG. 65 is an amino acid sequence of mature EPO, i.e. 165 amino acids(SEQ ID NO:73).

FIG. 66, comprising FIGS. 66A and 66B, is an exemplary nucleotide andcorresponding amino acid sequence of granulocyte-macrophage colonystimulating factor (GM-CSF) (SEQ ID NOS: 17 and 18, respectively).

FIG. 67, comprising FIGS. 67A and 67B, is an exemplary nucleotide andcorresponding amino acid sequence of interferon gamma (IFN-gamma) (SEQID NOS: 19 and 20, respectively).

FIG. 68, comprising FIGS. 68A and 68B, is an exemplary nucleotide andcorresponding amino acid sequence of α-1-protease inhibitor (A-1-PI, orα-antitrypsin) (SEQ ID NOS: 21 and 22, respectively).

FIG. 69, comprising FIGS. 69A-1 to 69A-2, and 69B, is an exemplarynucleotide and corresponding amino acid sequence of glucocerebrosidase(SEQ ID NOS: 23 and 24, respectively).

FIG. 70, comprising FIGS. 70A and 70B, is an exemplary nucleotide andcorresponding amino acid sequence of tissue-type plasminogen activator(TPA) (SEQ ID NOS: 25 and 26, respectively).

FIG. 71, comprising FIGS. 71A and 71B, is an exemplary nucleotide andcorresponding amino acid sequence of Interleukin-2 (IL-2) (SEQ ID NOS:27 and 28, respectively).

FIG. 72, comprising FIGS. 72A-1 through 72A-4 and FIG. 72B-1 through72B-4, is an exemplary nucleotide and corresponding amino acid sequenceof Factor VIII (SEQ ID NOS: 29 and 30, respectively).

FIG. 73, comprising FIGS. 73A and 73B, is an exemplary nucleotide andcorresponding amino acid sequence of urokinase (SEQ ID NOS: 33 and 34,respectively).

FIG. 74, comprising FIGS. 74A and 74B, is an exemplary nucleotide andcorresponding amino acid sequence of human recombinant DNase (hrDNase)(SEQ ID NOS: 39 and 40, respectively).

FIG. 75, comprising FIGS. 75A and 75B, is an exemplary nucleotide andcorresponding amino acid sequence of an insulin molecule (SEQ ID NOS: 43and 44, respectively).

FIG. 76, comprising FIGS. 76A and 76B, is an exemplary nucleotide andcorresponding amino acid sequence of S-protein from a Hepatitis B virus(HbsAg) (SEQ ID NOS: 45 and 46, respectively).

FIG. 77, comprising FIGS. 77A and 77B, is an exemplary nucleotide andcorresponding amino acid sequence of human growth hormone (hGH) (SEQ IDNOS: 47 and 48, respectively).

FIG. 78, comprising FIGS. 78A and 78B, are exemplary nucleotide andcorresponding amino acid sequences of anti-thrombin III. FIGS. 78A and78B, are an exemplary nucleotide and corresponding amino acid sequencesof “WT” anti-thrombin III (SEQ ID NOS: 63 and 64, respectively).

FIG. 79, comprising FIGS. 79A to 79D, are exemplary nucleotide andcorresponding amino acid sequences of human chorionic gonadotropin (hCG)α and β subunits. FIGS. 79A and 79B are an exemplary nucleotide andcorresponding amino acid sequence of the α-subunit of human chorionicgonadotropin (SEQ ID NOS: 69 and 70, respectively). FIGS. 79C and 79Dare an exemplary nucleotide and corresponding amino acid sequence of thebeta subunit of human chorionic gonadotrophin (SEQ ID NOS: 71 and 72,respectively).

FIG. 80, comprising FIGS. 80A and 80B, is an exemplary nucleotide andcorresponding amino acid sequence of α-iduronidase (SEQ ID NOS: 65 and66, respectively).

FIG. 81, comprising FIGS. 81A and 81B, is an exemplary nucleotide andcorresponding amino acid sequence of α-galactosidase A (SEQ ID NOS: 67and 68, respectively).

FIG. 82, comprising FIGS. 82A and 82B, is an exemplary nucleotide andcorresponding amino acid sequence of the 75 kDa tumor necrosis factorreceptor (TNF-R), which comprises a portion of Enbrel™ (tumor necrosisfactor receptor (TNF-R)/IgG fusion) (SEQ ID NOS: 31 and 32,respectively).

FIG. 83, comprising FIGS. 83A and 83B, is an exemplary amino acidsequence of the light and heavy chains, respectively, of Herceptin™(monoclonal antibody (MAb) to Her-2, human epidermal growth factorreceptor) (SEQ ID NOS: 35 and 36, respectively).

FIG. 84, comprising FIGS. 84A and 84B, is an exemplary amino acidsequence the heavy and light chains, respectively, of Synagis™ (MAb to Fpeptide of Respiratory Syncytial Virus) (SEQ ID NOS: 37 and 38,respectively).

FIG. 85, comprising FIGS. 85A and 85B, is an exemplary nucleotide andcorresponding amino acid sequence of the non-human variable regions ofRemicade™ (MAb to TNFα) (SEQ ID NOS: 41 and 42, respectively).

FIG. 86, comprising FIGS. 86A and 86B, is an exemplary nucleotide andcorresponding amino acid sequence of the Fc portion of human IgG (SEQ IDNOS: 49 and 50, respectively).

FIG. 87 is an exemplary amino acid sequence of the mature variableregion light chain of an anti-glycoprotein IIb/IIIa murine antibody (SEQID NO: 52).

FIG. 88 is an exemplary amino acid sequence of the mature variableregion heavy chain of an anti-glycoprotein IIb/IIIa murine antibody (SEQID NO: 54).

FIG. 89 is an exemplary amino acid sequence of variable region lightchain of a human IgG (SEQ ID NO: 51).

FIG. 90 is an exemplary amino acid sequence of variable region heavychain of a human IgG (SEQ ID NO:53).

FIG. 91 is an exemplary amino acid sequence of a light chain of a humanIgG (SEQ ID NO:55).

FIG. 92 is an exemplary amino acid sequence of a heavy chain of a humanIgG (SEQ ID NO:56).

FIG. 93, comprising FIGS. 93A and 93B, is an exemplary nucleotide andcorresponding amino acid sequence of the mature variable region of thelight chain of an anti-CD20 murine antibody (SEQ ID NOS: 59 and 60,respectively).

FIG. 94, comprising FIGS. 94A and 94B, is an exemplary nucleotide andcorresponding amino acid sequence of the mature variable region of theheavy chain of an anti-CD20 murine antibody (SEQ ID NOS: 61 and 62,respectively).

FIG. 95, comprising FIGS. 95A through, 95E, is the nucleotide sequenceof the tandem chimeric antibody expression vector TCAE 8 (SEQ ID NO:57).

FIG. 96, comprising FIGS. 96A through 96 E, is the nucleotide sequenceof the tandem chimeric antibody expression vector TCAE 8 containing thelight and heavy variable domains of the anti-CD20 murine antibody (SEQID NO:58).

FIG. 97, comprising FIGS. 97A to 97C, are graphs depicting 2-AA HPLCanalysis of glycans released by PNGaseF from myeloma-expressed Cri-IgG1antibody. The structure of the glycans is determined by retention time:the G0 glycoform elutes at 30 min., the G1 glycoform elutes at ˜33 min.,the G2 glycoform elutes at about approximately 37 min. and the S1-G1glycoform elutes at 70 min. FIG. 97A depicts the analysis of the DEAEantibody sample. FIG. 97B depicts the analysis of the SPA antibodysample. FIG. 97C depicts the analysis of the Fc antibody sample. Thepercent area under the peaks for these graphs is summarized in Table 14.

FIG. 98, comprising FIGS. 98A to 98C, are graphs depicting the MALDIanalysis of glycans released by PNGaseF from myeloma-expressed Cri-IgG1antibody. The glycans were derivatized with 2-AA and then analyzed byMALDI. FIG. 98A depicts the analysis of the DEAE antibody sample. FIG.98B depicts the analysis of the SPA antibody sample. FIG. 98C depictsthe analysis of the Fc antibody sample.

FIG. 99, comprising FIGS. 99A to 99D, are graphs depicting the capillaryelectrophoresis analysis of glycans released from Cri-IgG1 antibodiesthat have been glycoremodeled to contain M3N2 glycoforms. A graphdepicting the capillary electrophoresis analysis of glycan standardsderivatized with APTS is shown in FIG. 99A. FIG. 99B depicts theanalysis of the DEAE antibody sample. FIG. 99C depicts the analysis ofthe SPA antibody sample. FIG. 99D depicts the analysis of the Fcantibody sample. The percent area under the peaks for these graphs issummarized in Table 15.

FIG. 100, comprising FIGS. 100A to 100D, are graphs depicting thecapillary electrophoresis analysis of glycans released from Cri-IgG1antibodies that have been glycoremodeled to contain G0 glycoforms. Agraph depicting the capillary electrophoresis analysis of glycanstandards derivatized with APTS is shown in FIG. 100A. FIG. 100B depictsthe analysis of the DEAE antibody sample. FIG. 100C depicts the analysisof the SPA antibody sample. FIG. 100D depicts the analysis of the Fcantibody sample. The percent area under the peaks for these graphs issummarized in Table 16.

FIG. 101, comprising FIGS. 101A to 101C, are graphs depicting 2-AA HPLCanalysis of glycans released from Cri-IgG1 antibodies that have beenglycoremodeled to contain G0 glycoforms. The released glycans werelabeled with 2AA and separated by HPLC on a NH2P-50 4D amino column.FIG. 101A depicts the analysis of the DEAE antibody sample. FIG. 101Bdepicts the analysis of the SPA antibody sample. FIG. 101C depicts theanalysis of the Fc antibody sample. The percent area under the peaks forthese graphs is summarized in Table 16

FIG. 102, comprising FIGS. 102A to 102C, are graphs depicting the MALDIanalysis of glyeans released from Cri-IgG1 antibodies that have beenglycoremodeled to contain G0 glycoforms. The released glycans werederivatized with 2-AA and then analyzed by MALDI. FIG. 102A depicts theanalysis of the DEAE antibody sample. FIG. 102B depicts the analysis ofthe SPA antibody sample. FIG. 102C depicts the analysis of the Fcantibody sample.

FIG. 103, comprising FIGS. 103A to 103D, are graphs depicting thecapillary electrophoresis analysis of glycans released from Cri-IgG1antibodies that have been glycoremodeled to contain G2 glycoforms. Agraph depicting the capillary electrophoresis analysis of glycanstandards derivatized with APTS is shown in FIG. 103A. FIG. 103B depictsthe analysis of the DEAE antibody sample. FIG. 103C depicts the analysisof the SPA antibody sample. FIG. 103D depicts the analysis of the Fcantibody sample. The percent area under the peaks for these graphs issummarized in Table 17.

FIG. 104, comprising FIGS. 104A to 104C, are graphs depicting the 2-AAHPLC analysis of glycans released from remodeled Cri-IgG1 antibodiesthat have been glycoremodeled to contain G2 glycoforms. The releasedglycans were labeled with 2AA and then separated by HPLC on a NH2P-50 4Damino column. FIG. 104A depicts the analysis of the DEAE antibodysample. FIG. 104B depicts the analysis of the SPA antibody sample. FIG.104C depicts the analysis of the Fc antibody sample. The percent areaunder the peaks for these graphs is summarized in Table 17.

FIG. 105, comprising FIGS. 105A to 105C, are graphs depicting MALDIanalysis of glycans released from Cri-IgG1 antibodies that have beenglycoremodeled to contain G2 glycoforms. The released glycans werederivatized with 2-AA and then analyzed by MALDI. FIG. 105A depicts theanalysis of the DEAE antibody sample. FIG. 105B depicts the analysis ofthe SPA antibody sample. FIG. 105C depicts the analysis of the Fcantibody sample.

FIG. 106, comprising FIGS. 106A to 106D, are graphs depicting capillaryelectrophoresis analysis of glycans released from Cri-IgG1 antibodiesthat have been glycoremodeled by GnT-I treatment of M3N2 glycoforms. Agraph depicting the capillary electrophoresis analysis of glycanstandards derivatized with APTS is shown in FIG. 106A. FIG. 106B depictsthe analysis of the DEAE antibody sample. FIG. 106C depicts the analysisof the SPA antibody sample. FIG. 106D depicts the analysis of the Fcantibody sample.

FIG. 107, comprising FIGS. 107A to 107C, are graphs depicting 2-AA HPLCanalysis of glyeans released from Cri-IgG1 antibodies that have beenremodeled by GnT-I treatment of M3N2 glycoforms. The released glycanswere labeled with 2-AA and separated by HPLC on a NH2P-50 4D aminocolumn. FIG. 107A depicts the analysis of the DEAE antibody sample. FIG.107B depicts the analysis of the SPA antibody sample. FIG. 107C depictsthe analysis of the Fc antibody sample.

FIG. 108, comprising FIGS. 108A to 108C, are graphs depicting MALDIanalysis of glycans released from Cri-IgG1 antibodies that have beenglycoremodeled by GnT-I treatment of M3N2 glycoforms. The releasedglycans were derivatized with 2-AA and then analyzed by MALDI. FIG. 108Adepicts the analysis of the DEAE antibody sample. FIG. 108B depicts theanalysis of the SPA antibody sample. FIG. 108C depicts the analysis ofthe Fc antibody sample.

FIG. 109, comprising FIGS. 109A to 109D, are graphs depicting capillaryelectrophoresis of glycans released from Cri-IgG1 antibodies that havebeen glycoremodeled by GnT-I, II and III treatment of M3N2 glycoforms. Agraph depicting the capillary electrophoresis analysis of glycanstandards derivatized with APTS is shown in FIG. 109A. FIG. 109B depictsthe analysis of the DEAE antibody sample. FIG. 109C depicts the analysisof the SPA antibody sample. FIG. 109D depicts the analysis of the Fcantibody sample. The percent area under the peaks for these graphs issummarized in Table 18.

FIG. 110, comprising FIGS. 110A to 110C, are graphs depicting 2-AA HPLCanalysis of glycans released from Cri-IgG1 antibodies that have beenglycoremodeled by GnT-I, II and III treatment of M3N2 glycoforms. Thereleased glycans were labeled with 2AA and then separated by HPLC on aNH2P-50 4D amino column. FIG. 110A depicts the analysis of the DEAEantibody sample. FIG. 110B depicts the analysis of the SPA antibodysample. FIG. 110C depicts the analysis of the Fc antibody sample. Thepercent area under the peaks for these graphs is summarized in Table 18.

FIG. 111, comprising FIGS. 111A to 111C, are graphs depicting MALDIanalysis of glycans released from Cri-IgG1 antibodies that have beenglycoremodeled by galactosyltransferase treatment of NGA2F glycoforms.The released glycans were derivatized with 2-AA and then analyzed byMALDI. FIG. 111A depicts the analysis of the DEAE antibody sample. FIG.111B depicts the analysis of the SPA antibody sample. FIG. 111C depictsthe analysis of the Fc antibody sample.

FIG. 112, comprising 112A to 112D, are graphs depicting 2-AA HPLCanalysis of glycans released from Cri-IgG1 antibodies containing NGA2Fisoforms before GalT1 treatment (FIGS. 112A and 112C) and after GalT1treatment (FIGS. 112B and 112D). FIGS. 112A and 112B depict the analysisof the DEAE sample of antibodies. FIGS. 112C and 112D depict theanalysis of the Fe sample of antibodies. The released glycans werelabeled with 2AA and separated by HPLC on a NH2P-50 4D amino column.

FIG. 113, comprising 113A to 113C, are graphs depicting 2-AA HPLCanalysis of glycans released from Cri-IgG1 antibodies that have beenglycoremodeled by ST3Gal3 treatment of G2 glycoforms. The releasedglycans are labeled with 2-AA and then separated by HPLC on a NH2P-50 4Damino column. FIG. 113A depicts the analysis of the DEAE antibodysample. FIG. 113B depicts the analysis of the SPA antibody sample. FIG.113C depicts the analysis of the Fc antibody sample. The percent areaunder the peaks for these graphs is summarized in Table 19.

FIG. 114, comprising FIGS. 114A to 114C, are graphs depicting MALDIanalysis of glycans released from Cri-IgG1 antibodies that had beenglycoremodeled by ST3Gal3 treatment of G2 glycoforms. The releasedglycans were derivatized with 2-AA and then analyzed by MALDI. FIG. 114Adepicts the analysis of the DEAE antibody sample. FIG. 114B depicts theanalysis of the SPA antibody sample. FIG. 114C depicts the analysis ofthe Fc antibody sample.

FIG. 115, comprising FIGS. 115A to 115D, are graphs depicting capillaryelectrophoresis analysis of glycans released from Cri-IgG1 antibodiesthat had been glycoremodeled by ST6Gal1 treatment of G2 glycoforms. Agraph depicting the capillary electrophoresis analysis of glycanstandards derivatized with APTS is shown in FIG. 115A. FIG. 115B depictsthe analysis of the DEAE antibody sample. FIG. 115C depicts the analysisof the SPA antibody sample. FIG. 115D depicts the analysis of the Fcantibody sample.

FIG. 116, comprising FIGS. 116A to 116C, are graphs depicting 2-AA HPLCanalysis of glycans released from Cri-IgG1 antibodies that had beenglycoremodeled by ST6Gal1 treatment of G2 glycoforms. The releasedglycans were labeled with 2-AA and separated by HPLC on a NH2P-50 4Damino column. FIG. 116A depicts the analysis of the DEAE antibodysample. FIG. 116B depicts the analysis of the SPA antibody sample. FIG.116C depicts the analysis of the Fc antibody sample.

FIG. 117, comprising FIGS. 117A to 117C, are graphs depicting MALDIanalysis of glycans released from Cri-IgG1 antibodies that had beenglycoremodeled by ST6Gal1 treatment of G2 glycoforms. The releasedglycans were derivatized with 2-AA and then analyzed by MALDI. FIG. 117Adepicts the analysis of the DEAE antibody sample. FIG. 117B depicts theanalysis of the SPA antibody sample. FIG. 117C depicts the analysis ofthe Fc antibody sample.

FIG. 118, comprising FIGS. 118A to 118E, depicts images of SDS-PAGEanalysis of the glycoremodeled of Cri-IgG1 antibodies with differentglycoforms under non-reducing conditions. Bovine serum albumin (BSA) wasrun under reducing conditions as a quantitative standard. Proteinmolecular weight standards are displayed and their size is indicated inkDa. FIG. 11 8A depicts SDS-PAGE analysis of the DEAE, SPA and FcCri-IgG1 antibodies glycoremodeled to contain G0 and G2 glycoforms. FIG.118B depicts SDS-PAGE analysis of the DEAE, SPA and Fc Cri-IgG1antibodies glycoremodeled to contain NGA2F (bisecting) and GnT-I-M3N2(GnT1) glycoforms. FIG. 118C depicts SDS-PAGE analysis of the DEAE, SPAand Fc Cri-IgG1 antibodies glycoremodeled to contain S2G2 (ST6Gal1)glycoforms. FIG. 118D depicts SDS-PAGE analysis of the DEAE, SPA and FcCri-IgG1 antibodies glycoremodeled to contain M3N2 glycoforms, and BSA.FIG. 118E depicts SDS-PAGE analysis of the DEAE, SPA and Fc Cri-IgG1antibodies glycoremodeled to contain Gal-NGA2F (Gal-bisecting)glycoforms, and BSA.

FIG. 119 is an image of an acrylamide gel depicting the results of FACEanalysis of the pre- and post-sialylation of TP10. The BiNA₀ species hasno sialic acid residues. The BiNA₁ species has one sialic acid residue.The BiNA₂ species has two sialic acid residues. Bi=biantennary;NA=neuraminic acid.

FIG. 120 is a graph depicting the plasma concentration in μg/ml overtime of pre- and post-sialylation TP10 injected into rats.

FIG. 121 is a graph depicting the area under the plasmaconcentration-time curve (AUC) in μg/hr/ml for pre- and post sialylatedTP10.

FIG. 122 is an image of an acrylamide gel depicting the results of FACEglycan analysis of the pre- and post-fucosylation of TP10 and FACEglycan analysis of CHO cell produced TP-20. The BiNA₂F₂ species has twoneuraminic acid (NA) residues and two fucose residues (F).

FIG. 123 is a graph depicting the in vitro binding of TP20 (sCRIsLe^(X))glycosylated in vitro (diamonds) and in vivo in Lec11 CHO cells(squares).

FIG. 124 is a graph depicting the analysis by 2-AA HPLC of glycoformsfrom the GlcNAc-ylation of EPO.

FIG. 125, comprising FIGS. 125A and 125B, are graphs depicting the 2-AAHPLC analysis of two lots of EPO to which N-acetylglucosamine was beenadded. FIG. 125A depicts the analysis of lot A, and FIG. 125B depictsthe analysis of lot B.

FIG. 126 is a graph depicting the 2-AA HPLC analysis of the products thereaction introducing a third glycan branch to EPO with GnT-V.

FIG. 127 is a graph depicting a MALDI-TOF spectrum of the glycans of theEPO preparation after treatment with GnT-I, GnT-II, GnT-III, GnT-V andGalT1, with appropriate donor groups.

FIG. 128 is a graph depicting a MALDI spectrum the glycans of nativeEPO.

FIG. 129 is an image of an SDS-PAGE gel of the products of thePEGylation reactions using CMP-SA-PEG (1 kDa), and CMP-SA-PEG (10 kDa).

FIG. 130 is a graph depicting the results of the in vitro bioassay ofPEGylated EPO. Diamonds represent the data from sialylated EPO having noPEG molecules. Squares represent the data obtained using EPO with PEG (1kDa). Triangles represent the data obtained using EPO with PEG (10 kDa).

FIG. 131 is a diagram of CHO-expressed EPO. The EPO polypeptide is 165amino acids in length, with a molecular weight of 18 kDa withoutglycosylation. The glycosylated forms of EPO produced in CHO cells havea molecular weight of about 33 kDa to 39 kDa. The shapes which representthe sugars in the glycan chains are identified in the box at the loweredge of the drawing.

FIG. 132 is a diagram of insect cell expressed EPO. The shapes thatrepresent the sugars in the glycan chains are identified in the box atthe lower edge of FIG. 131.

FIG. 133 is a bar graph depicting the molecular weights of the EPOpeptides expressed in insect cells which were remodeled to form completemono-, bi- and tri-antennary glycans, with optional glycoPEGylation with1 kDa, 10 kDa or 20 kDa PEG. Epoetin™ is EPO expressed in mammaliancells without further glycan modification or PEGylation. NESP (Aranesp™,Amgen, Thousand Oaks, Calif.) is a form of EPO having 5 N-linked glycansites that is also expressed in mammalian cells without further glycanmodification or PEGylation.

FIG. 134, comprising FIGS. 134A and 134B, depicts one scheme for theremodeling and glycoPEGylation of insect cell expressed EPO. FIG. 134Adepicts the remodeling and glycoPEGylation steps that remodel the insectexpressed glycan to a mono-antennary glycoPEGylated glycan. FIG. 134Bdepicts the remodeled EPO polypeptide having a completed glycoPEGylatedmono-antennary glycan at each N-linked glycan site of the polypeptide.The shapes that represent the sugars in the glycan chains are identifiedin the box at the lower edge of FIG. 131, except that the trianglerepresents sialic acid.

FIG. 135 is a graph depicting the in vitro bioactivities of EPO-SA andEPO-SA-PEG constructs. The in vitro assay measured the proliferation ofTF-1 erythroleukemia cells which were maintained for 48 hr in RBMI+FBS10%+GM-CSF (12 ng/ml) after the EPO construct was added at 10.0, 5.0,2.0, 1.0, 0.5, and 0 μg/ml. Tri-SA refers to EPO constructs where theglycans are tri-antennary and have SA. Tri-SA 1K PEG refers to EPOconstructs where the glycans are tri-antennary and have Gal and are thenglycoPEGylated with SA-PEG 1 kDa. Di-SA 10K PEG refers to EPO constructswhere the glycans are bi-antennary and have Gal and are thenglycoPEGylated with SA-PEG 10 kDa. Di-SA 1K PEG refers to EPO constructswhere the glycans are bi-antennary and have Gal and are thenglycoPEGylated with SA-PEG 1 kDa. Di-SA refers to EPO constructs wherethe glycans are bi-antennary and are built out to SA. Epogen™ is EPOexpressed in CHO cells with no further glycan modification.

FIG. 136 is a graph depicting the pharmacokinetics of the EPO constructsin rat. Rats were bolus injected with [I¹²⁵]-labeled glycoPEGylated andnon-glycoPEGylated EPO. The graph shows the concentration of theradio-labeled EPO in the bloodstream of the rat at 0 to about 72 minutesafter injection. “Biant-10K” refers to EPO with biantennary glycanstructures with terminal 10 kDa PEG moieties. “Mono-20K” refers to EPOwith monoantennary glycan structures with terminal 20 kDa PEG moieties.NESP refers to the commercially available Aranesp. “Biant-1K” refers toEPO with biantennary glycan structures with terminal 1 kDa PEG moieties.“Biant-SA” refers to EPO with biantennary glycan structures withterminal 1 kDa moieties. The concentration of the EPO constructs in thebloodstream at 72 hr. is as follows: Biant-10K, 5.1 cpm/ml; Mono-20K,3.2 cpm/ml; NESP, 1 cpm/ml; and Biant-1K, 0.2 cpm/ml; Biant-SA, 0.1cpm/ml. The relative area under the curve of the EPO constructs is asfollows: Biant-10K, 2.9; Mono-20K, 2.1; NESP, 1; Biant-1K, 0.5; andBiant-SA, 0.2.

FIG. 137 is a bar graph depicting the ability of the EPO constructs tostimulate reticulocytosis in vivo. Each treatment group is composed ofeight mice. Mice were given a single subcutaneous injection of 10 μgprotein/kg body weight. The percent reticulocytosis was measured at 96hr. Tri-antennary-SA2,3(6) construct has the SA molecule bonded in a 2,3or 2,6 linkage (see, Example 18 herein for preparation) wherein theglycan on EPO is tri-antennary N-glycans with SA-PEG 10 K is attachedthereon. Similarly, bi-antennary-10K PEG is EPO having a bi-antennaryN-glycan with SA-PEG at 10 K PEG attached thereon.

FIG. 138 is a bar graph depicting the ability of EPO constructs toincrease the hematocrit of the blood of mice in vivo. CD-1 female micewere injected i.p. with 2.5 μg protein/kg body weight. The hematocrit ofthe mice was measured on day 15 after the EPO injection. Bi-1k refers toEPO constructs where the glycans are bi-antennary and are built out tothe Gal and then glycoPEGylated with SA-PEG 1 kDa. Mono-20k refers toEPO constructs where the glycans are mono-antennary and are built out tothe Gal and then glycoPEGylated with SA-PEG 20 kDa.

FIG. 139, comprising FIGS. 139A and 139B, depicts the analysis ofglycans enzymatically released from EPO expressed in insect cells(Protein Sciences, Lot #060302). FIG. 139A depicts the HPLC analysis ofthe released glycans. FIG. 139B depicts the MALDI analysis of thereleased glycans. Diamonds represent fucose, and squares representGlcNAc, circles represent mannose.

FIG. 140 depicts the MALDI analysis of glycans released from EPO afterthe GnT-I/GalT-1 reaction. The structures of the glycans have beendetermined by comparison of the peak spectrum with that of standardglycans. The glycan structures are depicted beside the peaks. Diamondsrepresent fucose, and squares represent GlcNAc, circles representmannose, stars represent galactose.

FIG. 141 depicts the SDS-PAGE analysis of EPO after the GnT-I/GalT-1reaction, Superdex 75 purification, ST3Gal3 reaction with SA-PEG (10kDa) and SA-PEG (20 kDa).

FIG. 142 depicts the results of the TF-1 cell in vitro bioassay ofPEGylated mono-antennary EPO.

FIG. 143, comprising FIGS. 143A and 143B, depicts the analysis of glycanreleased from EPO after the GnT-I/GnT-II reaction. FIG. 143A depicts theHPLC analysis of the released glycans, where peak 3 represents thebi-antennary GlcNAc glycan. FIG. 143B depicts the MALDI analysis of thereleased glycans. The structures of the glycans have been determined bycomparison of the peak spectrum with that of standard glycans. Theglycan structures are depicted beside the peaks. Diamonds representfucose, and squares represent GlcNAc, circles represent mannose.

FIG. 144, comprising FIGS. 144A and 144B, depict the HPLC analysis ofglycans released from EPO after the GalT-1 reaction. FIG. 144A depictsthe glycans released after the small scale GalT-1 reaction. FIG. 144Bdepicts the glycans released after the large scale GalT-1 reaction. Inboth figures, Peak 1 is the bi-antennary glycan with terminal galactosemoieties and Peak 2 is the bi-antennary glycan without terminalgalactose moieties.

FIG. 145 depicts the Superdex 75 chromatography separation of EPOspecies after the GalT-1 reaction. Peak 2 contains EPO with bi-antennaryglycans with terminal galactose moieties.

FIG. 146 depicts the SDS-PAGE analysis of each of the products of theglycoremodeling process to make bi-antennary glycans with terminalgalactose moieties.

FIG. 147 depicts the SDS-PAGE analysis of EPO after ST3Gal3 sialylationor PEGylation with SA-PEG (1 kDa) or SA-PEG (10 kDa).

FIG. 148 depicts the HPLC analysis of glycans released from EPO afterthe GnT-I/GnT-II reaction. The structures of the glycans have beendetermined by comparison of the peak retention with that of standardglycans. The glycan structures are depicted beside the peaks. Diamondsrepresent fucose, and squares represent GlcNAc, circles representmannose.

FIG. 149 depicts the HPLC analysis of glycans released from EPO afterthe GnT-V reaction. The structures of the glycans have been determinedby comparison of the peak retention with that of standard glycans. Theglycan structures are depicted beside the peaks. Diamonds representfucose, and squares represent GlcNAc, circles represent mannose.

FIG. 150 depicts the HPLC analysis of glycans released from EPO afterthe GalT-1 reaction. The structures of the glycans have been determinedby comparison of the peak retention with that of standard glyeans. Theglycan structures are depicted beside the peaks. Diamonds representfucose, and squares represent GlcNAc, circles represent mannose, opencircles represent galactose and triangles represent sialic acid.

FIG. 151 depicts the HPLC analysis of glycans released from EPO afterthe ST3Gal3 reaction. The structures of the glycans have been determinedby comparison of the peak retention with that of standard glycans. Theglycan structures are depicted beside the peaks. Diamonds representfucose, and squares represent GlcNAc, circles represent mannose, opencircles represent galactose and triangles represent sialic acid.

FIG. 152 depicts the HPLC analysis of glycans released from EPO afterthe ST6Gal1 reaction. The structures of the glycans have been determinedby comparison of the peak retention with that of standard glycans. Theglycan structures are depicted beside the peaks.

FIG. 153 depicts the results of the TF-1 cells in vitro bioassay of EPOwith bi-antennary and triantennary glycans. “Di-SA” refers to EPO withbi-antennary glycans that terminate in sialic acid. “Di-SA 10K PEG”refers to EPO with bi-antennary glycans that terminate in sialic acidderivatized with PEG (10 kDa). “Di-SA 1K PEG” refers to EPO withbi-antennary glycans that terminate in sialic acid derivatized with PEG(1 kDa). “Tri-SA ST6+ST3” refers to EPO with tri-antennary glycansterminating in 2,6-SA capped with 2,3-SA. “Tri-SA ST3” refers to EPOwith tri-antennary glycans terminating in 2,3-SA.

FIG. 154 is an image of an IEF gel depicting the pI of the products ofthe desialylation procedure. Lanes 1 and 5 are IEF standards. Lane 2 isFactor IX protein. Lane 3 is rFactor IX protein. Lane 4 is thedesialylation reaction of rFactor IX protein at 20 hr.

FIG. 155 is an image of an SDS-PAGE gel depicting the molecular weightof Factor IX conjugated with either SA-PEG (1 kDa) or SA-PEG (10 kDa)after reaction with CMP-SA-PEG. Lanes 1 and 6 are SeeBlue +2 molecularweight standards. Lane 2 is rF-IX. Lane 3 is desialylated rF-IX. Lane 4is rFactor IX conjugated to SA-PEG (1 kDa). Lane 5 is rFactor IXconjugated to SA-PEG (10 kDa).

FIG. 156 is an image of an SDS-PAGE gel depicting the reaction productsof direct-sialylation of Factor-IX and sialic acid capping ofFactor-IX-SA-PEG. Lane 1 is protein standards, lane 2 is blank; lane 3is rFactor-IX; lane 4 is SA capped rFactor-IX-SA-PEG (10 kDa); lane 5 isrFactor-IX-SA-PEG (10 kDa); lane 6 is ST3Gall; lane 7 is ST3Gal3; lanes8, 9, 10 are rFactor-IX-SA-PEG(10 kDa) with no prior sialidasetreatment.

FIG. 157 is an image of an isoelectric focusing gel (pH 3-7) ofasialo-Factor VIIa. Lane 1 is rFactor VIIa; lanes 2-5 are asialo-FactorVIIa.

FIG. 158 is a graph of a MALDI spectra of Factor VIIa.

FIG. 159 is a graph of a MALDI spectra of Factor VIIa-PEG (1 kDa).

FIG. 160 is a graph depicting a MALDI spectra of Factor VIIa-PEG (10kDa).

FIG. 161 is an image of an SDS-PAGE gel of PEGylated Factor VIIa. Lane 1is asialo-Factor VIIa. Lane 2 is the product of the reaction ofasialo-Factor VIIa and CMP-SA-PEG(1 kDa) with ST3Gal3 after 48 hr. Lane3 is the product of the reaction of asialo-Factor VIIa and CMP-SA-PEG (1kDa) with ST3Gal3 after 48 hr. Lane 4 is the product of the reaction ofasialo-Factor VIIa and CMP-SA-PEG (10 kDa) with ST3Gal3 at 96 hr.

FIG. 162 is an image of an isoelectric focusing (IEF) gel depicting theproducts of the desialylation reaction of human pituitary FSH. Lanes 1and 4 are isoelectric focusing (IEF) standards. Lane 2 is native FSH.Lane 3 is desialylated FSH.

FIG. 163 is an image of an SDS-PAGE gel of the products of the reactionsto make PEG-sialylation of rFSH. Lanes 1 and 8 are SeeBlue+2 molecularweight standards. Lane 2 is 15 μg of native FSH. Lane 3 is 15 μg ofasialo-FSH (AS-FSH). Lane 4 is 15 μg of the products of the reaction ofAS-FSH with CMP-SA. Lane 5 is 15 μg of the products of the reaction ofAS-FSH with CMP-SA-PEG (1 kDa). Lane 6 is 15 μg of the products of thereaction of AS-FSH with CMP-SA-PEG (5 kDa). Lane 7 is 15 μg of theproducts of the reaction of AS-FSH with CMP-SA-PEG (10 kDa).

FIG. 164 is an image of an isoelectric focusing gel of the products ofthe reactions to make PEG-sialylation of FSH. Lanes 1 and 8 are IEFstandards. Lane 2 is 15 μg of native FSH. Lane 3 is 15 μg of asialo-FSH(AS-FSH). Lane 4 is 15 μg of the products of the reaction of AS-FSH withCMP-SA. Lane 5 is 15 μg of the products of the reaction of AS-FSH withCMP-SA-PEG (1 kDa). Lane 6 is 15 μg of the products of the reaction ofAS-FSH with CMP-SA-PEG (5 kDa). Lane 7 is 15 μg of the products of thereaction of AS-FSH with CMP-SA-PEG (10 kDa).

FIG. 165 is an image of an SDS-PAGE gel of native non-recombinant FSHproduced in human pituitary cells. Lanes 1, 2 and 5 are SeeBlue™+2molecular weight standards. Lanes 3 and 4 are native FSH at 5 μg and 25μg, respectively.

FIG. 166 is an image of an isoelectric focusing gel (pH 3-7) depictingthe products of the asialylation reaction of rFSH. Lanes 1 and 4 are IEFstandards. Lane 2 is native rFSH. Lane 3 is asialo-rFSH.

FIG. 167 is an image of an SDS-PAGE gel depicting the results of thePEG-sialylation of asialo-rFSH. Lane 1 is native rFSH. Lane 2 isasialo-FSH. Lane 3 is the products of the reaction of asialo-FSH andCMP-SA. Lanes 4-7 are the products of the reaction between asialoFSH and0.5 mM CMP-SA-PEG (10 kDa) at 2 hr, 5 hr, 24 hr, and 48 hr,respectively. Lane 8 is the products of the reaction between asialo-FSHand 1.0 mM CMP-SA-PEG (10 kDa) at 48 hr. Lane 9 is the products of thereaction between asialo-FSH and 1.0 mM CMP-SA-PEG (1 kDa) at 48 hr.

FIG. 168 is an image of an isoelectric focusing gel showing the productsof PEG-sialylation of asialo-rFSH with a CMP-SA-PEG (1 kDa). Lane 1 isnative rFSH. Lane 2 is asialo-rFSH. Lane 3 is the products of thereaction of asialo-rFSH and CMP-SA at 24 hr. Lanes 4-7 are the productsof the reaction of asialo-rFSH and 0.5 mM CMP-SA-PEG (1 kDa) at 2 hr, 5hr, 24 hr, and 48 hr, respectively. Lane 8 is blank. Lanes 9 and 10 arethe products of the reaction at 48 hr of asialo-rFSH and CMP-SA-PEG (10kDa) at 0.5 mM and 1.0 mM, respectively.

FIG. 169 is graph of the pharmacokinetics of rFSH and rFSH-SA-PEG (1 kDaand 10 kDa). This graph illustrates the relationship between the time arFSH compound is in the blood stream of the rat, and the meanconcentration of the rFSH compound in the blood for glycoPEGylated rFSHas compared to non-PEGylated rFSH.

FIG. 170 is a graph of the results of the FSH bioassay using Sertolicells. This graph illustrates the relationship between the FSHconcentration in the Sertoli cell incubation medium and the amount of17-β estradiol released from the Sertoli cells.

FIG. 171 is a graph depicting the results of the Steelman-Pohleybioassay of glycoPEGylated and non-glycoPEGylated FSH. Rats weresubcutaneously injected with human chorionic gonadotropin and varyingamounts of FSH for three days, and the average ovarian weight of thetreatment group determined on day 4. rFSH-SA-PEG refers to recombinantFSH that has been glycoPEGylated with PEG (1 kDa). rFSH refers tonon-glycoPEGylated FSH. Each treatment group contains 10 rats.

FIG. 172, comprising FIGS. 172A and 172B, depicts the chromatogram ofINF-β elution from a Superdex-75 column. FIG. 172A depicts the entirechromatogram. FIG. 172B depicts the boxed area of FIG. 172A containingpeaks 4 and 5 in greater detail.

FIG. 173, comprising FIGS. 173A and 173B, depict MALDI analysis ofglycans enzymatically released from INF-β. FIG. 173A depicts the MALDIanalysis glycans released from native INF-β. FIG. 173B depicts the MALDIanalysis of glycans released from desialylated INF-β. The structures ofthe glycans have been determined by comparison of the peak spectrum withthat of standard glycans. The glycan structures are depicted beside thepeaks. Squares represent GlcNAc, triangles represent fucose, circlesrepresent mannose, diamonds represent galactose and stars representsialic acid.

FIG. 174 depicts the lectin blot analysis of the sialylation of thedesialylated INF-P. The blot on the right side is detected with Maackiaamurensis agglutinin (MAA) labeled with digoxogenin (DIG) (Roche AppliedScience, Indianapolis, Ill.) to detect α2,3-sialylation. The blot on theleft is detected with Erthrina cristagalli lectin (ECL) labeled withbiotin (Vector Laboratories, Burlingame, Calif.) to detect exposedgalactose residues.

FIG. 175 depicts the SDS-PAGE analysis of the products of the PEG (10kDa) PEGylation reaction of INF-β. “-PEG” refers to INF-β before thePEGylation reaction. “+PEG” refers to INF-β after the PEGylationreaction.

FIG. 176 depicts the SDS-PAGE analysis of the products of the PEG (20kDa) PEGylation reaction of INF-β. “Unmodified” refers to INF-β beforethe PEGylation reaction. “Pegylated” refers to INF-β after thePEGylation reaction.

FIG. 177 depicts the chromatogram of PEG (10 kDa) PEGylated INF-βelution from a Superdex-200 column.

FIG. 178 depicts the results of a bioassay of peak fractions of PEG (10kDa) PEGylated INF-β shown in the chromatogram depicted Figure INF-PEG6.

FIG. 179 depicts the chromatogram of PEG (20 kDa) PEGylated INF-βelution from a Superdex-200 column.

FIG. 180, comprising FIGS. 180A and 180B, is two graphs depicting theMALDI-TOF spectrum of RNaseB (FIG. 180A) and the HPLC profile of theoligosaccharides cleaved from RNaseB by N-Glycanase (FIG. 180B). Themajority of N-glycosylation sites of the peptide are modified with highmannose oligosaccharides consisting of 5 to 9 mannose residues.

FIG. 181 is a scheme depicting the conversion of high mannose N-Glycansto hybrid N-Glycans. Enzyme 1 is α1,2-mannosidase, from Trichodomareesei or Aspergillus saitoi. Enzyme 2 is GnT-I (β-1,2-N-acetylglucosaminyl transferase I). Enzyme 3 is GalT-I(β1,4-galactosyltransfease 1). Enzyme 4 is α2,3-sialyltransferase orα2,6-sialyltransferase.

FIG. 182, comprising FIGS. 182A and 182B, is two graphs depicting theMALDI-TOF spectrum of RNaseB treated with a recombinant T. reeseiα1,2-mannosidase (FIG. 182A) and the HPLC profile of theoligosaccharides cleaved by N-Glycanase from the modified RNaseB (FIG.182B).

FIG. 183 is a graph depicting the MALDI-TOF spectrum of RNaseB treatedwith a commercially available α1,2-mannosidase purified from A. saitoi(Glyko & CalBioChem).

FIG. 184 is a graph depicting the MALDI-TOF spectrum of modified RNaseBby treating the product shown in FIG. 182 with a recombinant GnT-I(GlcNAc transferase-I).

FIG. 185 is a graph depicting the MALDI-TOF spectrum of modified RNaseBby treating the product shown in FIG. 184 with a recombinant GalT 1(galactosyltransferase 1).

FIG. 186 is a graph depicting the MALDI-TOF spectrum of modified RNaseBby treating the product shown in FIG. 185 with a recombinant ST3Gal III(α2,3-sialyltransferase III) using CMP-SA as the donor for thetransferase.

FIG. 187, comprising FIGS. 187A and 187B, are graphs depicting theMALDI-TOF spectrum of modified RNaseB by treating the product shown inFIG. 185 with a recombinant ST3Gal III (α2,3-sialyltransferase III)using CMP-SA-PEG (10 kDa) as the donor for the transferase.

FIG. 188 is a series of schemes depicting the conversion of high mannoseN-glycans to complex N-glycans. Enzyme 1 is α1,2-mannosidase fromTrichoderma reesei or Aspergillus saitoi. Enzyme 2 is GnT-I. Enzyme 3 isGAlT 1. Enzyme 4 is α2,3-sialyltransferase or α2,6-sialyltransferase.Enzyme 5 is α-mannosidase II. Enzyme 6 is α-mannosidase. Enzyme 7 isGnT-II. Enzyme 8 is α1,6-mannosidase. Enzyme 9 is α1,3-mannosidase.

FIG. 189 is a diagram of the linkage catalyzed byN-acetylglucosaminyltransferase I to VI (GnT I-VI).R=GlcNAcβ1,4GlcNAc-Asn-X.

FIG. 190 is an image of an SDS-PAGE gel: standard (Lane 1); nativetransferrin (Lane 2); asialotransferrin (Lane 3); asialotransferrin andCMP-SA (Lane 4); Lanes 5 and 6, asialotransferrin and CMP-SA-PEG (1 kDa)at 0.5 mM and 5 mM, respectively; Lanes 7 and 8, asialotransferrin andCMP-SA-PEG (5 kDa) at 0.5 mM and 5 mM, respectively; Lanes 9 and 10,asialotransferrin and CMP-SA-PEG (10 kDa) at 0.5 mM and 5 mM,respectively.

FIG. 191 is an image of an IEF gel: native transferrin (Lane 1);asialotransferrin (Lane 2); asialotaansferrin and CMP-SA, 24 hr (Lane3); asialotransferrin and CMP-SA, 96 hr (Lane 4) Lanes 5 and 6,asialotransferrin and CMP-SA-PEG (1 kDa) at 24 hr and 96 hr,respectively; Lanes 7 and 8, asialotransferrin and CMP-SA-PEG (5 kDa) at24 hr and 96 hr, respectively; Lanes 9 and 10, asialotransferrin andCMP-SA-PEG (10 kDa) at 24 hr and 96 hr, respectively.

FIG. 192 is a graph depicting the effects of EPO-SA-PEG (10 kDa),EPO-SA-PEG (20 kDa), and NESP on hemoglobin response in Sprague Dawleyrats. Rats were dosed (indicated by arrows) by subcutaneous injection of50 μg protein per Kg body weight. Values plotted are mean (n=5/group)increment in total blood hemoglobin baseline corrected against valuesobtained for control animals injected with vehicle alone.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes methods and compositions for the cellfree in vitro addition and/or deletion of sugars to or from a peptidemolecule in such a manner as to provide a glycopeptide molecule having aspecific customized or desired glycosylation pattern, wherein theglycopeptide is produced at an industrial scale. In a preferredembodiment of the invention, the glycopeptide so produced has attachedthereto a modified sugar that has been added to the peptide via anenzymatic reaction. A key feature of the invention is to take a peptideproduced by any cell type and generate a core glycan structure on thepeptide, following which the glycan structure is then remodeled in vitroto generate a glycopeptide having a glycosylation pattern suitable fortherapeutic use in a mammal. More specifically, it is possible accordingto the present invention, to prepare a glycopeptide molecule having amodified sugar molecule or other compound conjugated thereto, such thatthe conjugated molecule confers a beneficial property on the peptide.According to the present invention, the conjugate molecule is added tothe peptide enzymatically because enzyme-based addition of conjugatemolecules to peptides has the advantage of regioselectivity andstereoselectivity. The glycoconjugate may be added to the glycan on apeptide before or after glycosylation has been completed. In otherwords, the order of glycosylation with respect to glycoconjugation maybe varied as described elsewhere herein. It is therefore possible, usingthe methods and compositions provided herein, to remodel a peptide toconfer upon the peptide a desired glycan structure preferably having amodified sugar attached thereto. It is also possible, using the methodsand compositions of the invention to generate peptide molecules havingdesired and or modified glycan structures at an industrial scale,thereby, for the first time, providing the art with a practical solutionfor the efficient production of improved therapeutic peptides.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and nucleic acidchemistry and hybridization are those well known and commonly employedin the art. Standard techniques are used for nucleic acid and peptidesynthesis. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (e.g., Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, 2d ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.), which are provided throughout this document. The nomenclatureused herein and the laboratory procedures used in analytical chemistryand organic syntheses described below are those well known and commonlyemployed in the art. Standard techniques or modifications thereof, areused for chemical syntheses and chemical analyses.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as singlechain antibodies and humanized antibodies (Harlow et al., 1999, UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold SpringHarbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; Bird et al., 1988, Science 242:423-426).

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

As used herein, a “functional” biological molecule is a biologicalmolecule in a form in which it exhibits a property by which it ischaracterized. A functional enzyme, for example, is one which exhibitsthe characteristic catalytic activity by which the enzyme ischaracterized.

As used herein, the structure

is the point of connection between an amino acid or an amino acidsidechain in the peptide chain and the glycan structure.

“N-linked” oligosaccharides are those oligosaccharides that are linkedto a peptide backbone through asparagine, by way of anasparagine-N-acetylglucosamine linkage. N-linked oligosaccharides arealso called “N-glycans.” All N-linked oligosaccharides have a commonpentasaccharide core of Man₃GlcNAc₂. They differ in the presence of, andin the number of branches (also called antennae) of peripheral sugarssuch as N-acetylglucosamine, galactose, N-acetylgalactosamine, fucoseand sialic acid. Optionally, this structure may also contain a corefucose molecule and/or a xylose molecule.

An “elemental trimannosyl core structure” refers to a glycan moietycomprising solely a trimannosyl core structure, with no additionalsugars attached thereto. When the term “elemental” is not included inthe description of the “trimannosyl core structure,” then the glycancomprises the trimannosyl core structure with additional sugars attachedthereto. Optionally, this structure may also contain a core fucosemolecule and/or a xylose molecule.

The term “elemental trimannosyl core glycopeptide” is used herein torefer to a glycopeptide having glycan structures comprised primarily ofan elemental trimannosyl core structure. Optionally, this structure mayalso contain a core fucose molecule and/or a xylose Molecule.

“O-linked” oligosaccharides are those oligosaccharides that are linkedto a peptide backbone through threonine, serine, hydroxyproline,tyrosine, or other hydroxy-containing amino acids.

All oligosaccharides described herein are described with the name orabbreviation for the non-reducing saccharide (i.e., Gal), followed bythe configuration of the glycosidic bond (α or β), the ring bond (1 or2), the ring position of the reducing saccharide involved in the bond(2, 3, 4, 6 or 8), and then the name or abbreviation of the reducingsaccharide (i.e., GlcNAc). Each saccharide is preferably a pyranose. Fora review of standard glycobiology nomenclature see, Essentials ofGlycobiology Varki et al. eds., 1999, CSHL Press.

The term “sialic acid” refers to any member of a family of nine-carboncarboxylated sugars. The most common member of the sialic acid family isN-acetyl-neuraminic acid(2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onicacid (often abbreviated as NeuSAc, NeuAc, or NANA). A second member ofthe family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which theN-acetyl group of NeuAc is hydroxylated. A third sialic acid familymember is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J.Biol. Chem. 261: 11550-11557; Kanamori et al., J. Biol. Chem. 265:21811-21819 (1990)). Also included are 9-substituted sialic acids suchas a 9-O—C₁-C₆ acyl-NeuSAc like 9-O-lactyl-NeuSAc or 9-O-acetyl-Neu5Ac,9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of thesialic acid family, see, e.g., Varki, Glycobiology 2: 25-40 (1992);Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed.(Springer-Verlag, New York (1992)). The synthesis and use of sialic acidcompounds in a sialylation procedure is disclosed in internationalapplication WO 92/16640, published Oct. 1, 1992.

A peptide having “desired glycosylation”, as used herein, is a peptidethat comprises one or more oligosaccharide molecules which are requiredfor efficient biological activity of the peptide.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

The “area under the curve” or “AUC”, as used herein in the context ofadministering a peptide drug to a patient, is defined as total areaunder the curve that describes the concentration of drug in systemiccirculation in the patient as a function of time from zero to infinity.

The term “half-life” or “t ½”, as used herein in the context ofadministering a peptide drug to a patient, is defined as the timerequired for plasma concentration of a drug in a patient to be reducedby one half. There may be more than one half-life associated with thepeptide drug depending on multiple clearance mechanisms, redistribution,and other mechanisms well known in the art. Usually, alpha and betahalf-lives are defined such that the alpha phase is associated withredistribution, and the beta phase is associated with clearance.However, with protein drugs that are, for the most part, confined to thebloodstream, there can be at least two clearance half-lives. For someglycosylated peptides, rapid beta phase clearance may be mediated viareceptors on macrophages, or endothelial cells that recognize terminalgalactose, N-acetylgalactosamine, N-acetylglucosamine, mannose, orfucose. Slower beta phase clearance may occur via renal glomerularfiltration for molecules with an effective radius <2 nm (approximately68 kD) and/or specific or non-specific uptake and metabolism in tissues.GlycoPEGylation may cap terminal sugars (e.g. galactose orN-acetylgalactosamine) and thereby block rapid alpha phase clearance viareceptors that recognize these sugars. It may also confer a largereffective radius and thereby decrease the volume of distribution andtissue uptake, thereby prolonging the late beta phase. Thus, the preciseimpact of glycoPEGylation on alpha phase and beta phase half-lives willvary depending upon the size, state of glycosylation, and otherparameters, as is well known in the art. Further explanation of“half-life” is found in Pharmaceutical Biotechnology (1997, DFACrommelin and RD Sindelar, eds., Harwood Publishers, Amsterdam, pp101-120).

The term “residence time”, as used herein in the context ofadministering a peptide drug to a patient, is defined as the averagetime that drug stays in the body of the patient after dosing.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid nucleic acid encodingadditional peptide sequence.

A “polynucleotide” means a single strand or parallel and anti-parallelstrands of a nucleic acid. Thus, a polynucleotide may be either asingle-stranded or a double-stranded nucleic acid.

The term “nucleic acid” typically refers to large polynucleotides. Theterm “oligonucleotide” typically refers to short polynucleotides,generally no greater than about 50 nucleotides.

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction. Thedirection of 5′ to 3′ addition of nucleotides to nascent RNA transcriptsis referred to as the transcription direction. The DNA strand having thesame sequence as an mRNA is referred to as the “coding strand”;sequences on the DNA strand which are located 5′ to a reference point onthe DNA are referred to as “upstream sequences”; sequences on the DNAstrand which are 3′ to a reference point on the DNA are referred to as“downstream sequences.”

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a nucleic acid sequenceencodes a protein if transcription and translation of mRNA correspondingto that nucleic acid produces the protein in a cell or other biologicalsystem. Both the coding strand, the nucleotide sequence of which isidentical to the mRNA sequence and is usually provided in sequencelistings, and the non-coding strand, used as the template fortranscription of a gene or cDNA, can be referred to as encoding theprotein or other product of that nucleic acid or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopeptide molecules. When a subunit position in both of the two moleculesis occupied by the same monomeric subunit, e.g., if a position in eachof two DNA molecules is occupied by adenine, then they are homologous atthat position. The homology between two sequences is a direct functionof the number of matching or homologous positions, e.g., if half (e.g.,five positions in a polymer ten subunits in length) of the positions intwo compound sequences are homologous then the two sequences are 50%homologous, if 90% of the positions, e.g., 9 of 10, are matched orhomologous, the two sequences share 90% homology. By way of example, theDNA sequences 3′ATTGCC5′ and 3′TATGGC share 50% homology.

As used herein, “homology” is used synonymously with “identity.”

The determination of percent identity between two nucleotide or aminoacid sequences can be accomplished using a mathematical algorithm. Forexample, a mathematical algorithm useful for comparing two sequences isthe algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl.Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into theNBLAST and XBLAST programs of Altschul, et al. (1990, J. Mol. Biol.215:403-410), and can be accessed, for example at the National Centerfor Biotechnology Information (NCBI) world wide web site having theuniversal resource locator “http://www.ncbi.nlm.nih.gov/BLAST/”. BLASTnucleotide searches can be performed with the NBLAST program (designated“blastn” at the NCBI web site), using the following parameters: gappenalty=5; gap extension penalty=2; mismatch penalty=3; match reward=1;expectation value 10.0; and word size=11 to obtain nucleotide sequenceshomologous to a nucleic acid described herein. BLAST protein searchescan be performed with the XBLAST program (designated “blastn” at theNCBI web site) or the NCBI “blastp” program, using the followingparameters: expectation value 10.0, BLOSUM62 scoring matrix to obtainamino acid sequences homologous to a protein molecule described herein.To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al. (1997, Nucleic Acids Res.25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used toperform an iterated search which detects distant relationships betweenmolecules (Id.) and relationships between molecules which share a commonpattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

A “heterologous nucleic acid expression unit” encoding a peptide isdefined as a nucleic acid having a coding sequence for a peptide ofinterest operably linked to one or more expression control sequencessuch as promoters and/or repressor sequences wherein at least one of thesequences is heterologous, i.e., not normally found in the host cell.

By describing two polynucleotides as “operably linked” is meant that asingle-stranded or double-stranded nucleic acid moiety comprises the twopolynucleotides arranged within the nucleic acid moiety in such a mannerthat at least one of the two polynucleotides is able to exert aphysiological effect by which it is characterized upon the other. By wayof example, a promoter operably linked to the coding region of a nucleicacid is able to promote transcription of the coding region.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulator sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive promoter is a promoter which drives expression of a geneto which it is operably linked, in a constant manner in a cell. By wayof example, promoters which drive expression of cellular housekeepinggenes are considered to be constitutive promoters.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living cell substantiallyonly when an inducer which corresponds to the promoter is present in thecell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses that incorporate the recombinant polynucleotide.

A “genetically engineered” or “recombinant” cell is a cell having one ormore modifications to the genetic material of the cell. Suchmodifications are seen to include, but are not limited to, insertions ofgenetic material, deletions of genetic material and insertion of geneticmaterial that is extrachromasomal whether such material is stablymaintained or not.

A “peptide” is an oligopeptide, polypeptide, peptide, protein orglycoprotein. The use of the term “peptide” herein includes a peptidehaving a sugar molecule attached thereto when a sugar molecule isattached thereto.

As used herein, “native form” means the form of the peptide whenproduced by the cells and/or organisms in which it is found in nature.When the peptide is produced by a plurality of cells and/or organisms,the peptide may have a variety of native forms.

“Peptide” refers to a polymer in which the monomers are amino acids andare joined together through amide bonds, alternatively referred to as apeptide. Additionally, unnatural amino acids, for example, β-alanine,phenylglycine and homoarginine are also included. Amino acids that arenot nucleic acid-encoded may also be used in the present invention.Furthermore, amino acids that have been modified to include reactivegroups, glycosylation sites, polymers, therapeutic moieties,biomolecules and the like may also be used in the invention. All of theamino acids used in the present invention may be either the D- orL-isomer thereof. The L-isomer is generally preferred. In addition,other peptidomimetics are also useful in the present invention. As usedherein, “peptide” refers to both glycosylated and unglycosylatedpeptides. Also included are peptides that are incompletely glycosylatedby a system that expresses the peptide. For a general review, see,Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDESAND PROTEINS, B. Weinstein, eds., Marcel Dekker, New York, p. 267(1983).

The term “peptide conjugate,” refers to species of the invention inwhich a peptide is conjugated with a modified sugar as set forth herein.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is linked toa hydrogen, a carboxyl group, an amino group, and an A group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that function in amanner similar to a naturally occurring amino acid.

As used herein, amino acids are represented by the full name thereof, bythe three letter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated in the following Table 1:

TABLE 1 Amino acids, and the three letter and one letter codes. FullName Three-Letter Code One-Letter Code Aspartic Acid Asp D Glutamic AcidGlu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

The present invention also provides for analogs of proteins or peptideswhich comprise a protein as identified above. Analogs may differ fromnaturally occurring proteins or peptides by conservative amino acidsequence differences or by modifications which do not affect sequence,or by both. For example, conservative amino acid changes may be made,which although they alter the primary sequence of the protein orpeptide, do not normally alter its function. Conservative amino acidsubstitutions typically include substitutions within the followinggroups:

-   -   glycine, alanine;    -   valine, isoleucine, leucine;    -   aspartic acid, glutamic acid;    -   asparagine, glutamine;    -   serine, threonine;    -   lysine, arginine;    -   phenylalanine, tyrosine.

Modifications (which do not normally alter primary sequence) include invivo, or in vitro, chemical derivatization of peptides, e.g.,acetylation, or carboxylation. Also included are modifications ofglycosylation, e.g., those made by modifying the glycosylation patternsof a peptide during its synthesis and processing or in furtherprocessing steps; e.g., by exposing the peptide to enzymes which affectglycosylation, e.g., mammalian glycosylating or deglycosylating enzymes.Also embraced are sequences which have phosphorylated amino acidresidues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

It will be appreciated, of course, that the peptides may incorporateamino acid residues which are modified without affecting activity. Forexample, the termini may be derivatized to include blocking groups, i.e.chemical substituents suitable to protect and/or stabilize the N- andC-termini from “undesirable degradation”, a term meant to encompass anytype of enzymatic, chemical or biochemical breakdown of the compound atits termini which is likely to affect the function of the compound, i.e.sequential degradation of the compound at a terminal end thereof.

Blocking groups include protecting groups conventionally used in the artof peptide chemistry which will not adversely affect the in vivoactivities of the peptide. For example, suitable N-terminal blockinggroups can be introduced by alkylation or acylation of the N-terminus.Examples of suitable N-terminal blocking groups include C₁-C₅ branchedor unbranched alkyl groups, acyl groups such as formyl and acetylgroups, as well as substituted forms thereof, such as theacetamidomethyl (Acm), Fmoc or Boc groups. Desaamino analogs of aminoacids are also useful N-terminal blocking groups, and can either becoupled to the N-terminus of the peptide or used in place of theN-terminal reside. Suitable C-terminal blocking groups, in which thecarboxyl group of the C-terminus is either incorporated or not, includeesters, ketones or amides. Ester or ketone-forming alkyl groups,particularly lower alkyl groups such as methyl, ethyl and propyl, andamide-forming amino groups such as primary amines (—NH₂), and mono- anddi-alkylamino groups such as methylamino, ethylamino, dimethylamino,diethylamino, methylethylamino and the like are examples of C-terminalblocking groups. Descarboxylated amino acid analogues such as agmatineare also useful C-terminal blocking groups and can be either coupled tothe peptide's C-terminal residue or used in place of it. Further, itwill be appreciated that the free amino and carboxyl groups at thetermini can be removed altogether from the peptide to yield desamino anddescarboxylated forms thereof without affect on peptide activity.

Other modifications can also be incorporated without adversely affectingthe activity and these include, but are not limited to, substitution ofone or more of the amino acids in the natural L-isomeric form with aminoacids in the D-isomeric form. Thus, the peptide may include one or moreD-amino acid resides, or may comprise amino acids which are all in the.D-form. Retro-inverso forms of peptides in accordance with the presentinvention are also contemplated, for example, inverted peptides in whichall amino acids are substituted with D-amino acid forms.

Acid addition salts of the present invention are also contemplated asfunctional equivalents. Thus, a peptide in accordance with the presentinvention treated with an inorganic acid such as hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, and the like, or an organicacid such as an acetic, propionic, glycolic, pyruvic, oxalic, malic,malonic, succinic, maleic, fumaric, tataric, citric, benzoic, cinnamic,mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicyclicand the like, to provide a water soluble salt of the peptide is suitablefor use in the invention.

Also included are peptides which have been modified using ordinarymolecular biological techniques so as to improve their resistance toproteolytic degradation or to optimize solubility properties or torender them more suitable as a therapeutic agent. Analogs of suchpeptides include those containing residues other than naturallyoccurring L-amino acids, e.g., D-amino acids or non-naturally occurringsynthetic amino acids. The peptides of the invention are not limited toproducts of any of the specific exemplary processes listed herein.

As used herein, the term “MALDI” is an abbreviation for Matrix AssistedLaser Desorption Ionization. During ionization, SA-PEG (sialicacid-poly(ethylene glycol)) can be partially eliminated from theN-glycan structure of the glycoprotein.

As used herein, the term “glycosyltransferase,” refers to anyenzyme/protein that has the ability to transfer a donor sugar to anacceptor moiety.

As used herein, the term “modified sugar,” refers to a naturally- ornon-naturally-occurring carbohydrate that is enzymatically added onto anamino acid or a glycosyl residue of a peptide in a process of theinvention. The modified sugar is selected from a number of enzymesubstrates including, but not limited to sugar nucleotides (mono-, di-,and tri-phosphates), activated sugars (e.g., glycosyl halides, glycosylmesylates) and sugars that are neither activated nor nucleotides.

The “modified sugar” is covalently functionalized with a “modifyinggroup.” Useful modifying groups include, but are not limited to,water-soluble polymers, therapeutic moieties, diagnostic moieties,biomolecules and the like. The locus of functionalization with themodifying group is selected such that it does not prevent the “modifiedsugar” from being added enzymatically to a peptide.

The term “water-soluble” refers to moieties that have some detectabledegree of solubility in water. Methods to detect and/or quantify watersolubility are well known in the art. Exemplary water-soluble polymersinclude peptides, saccharides, poly(ethers), poly(amines),poly(carboxylic acids) and the like. Peptides can have mixed sequencesor be composed of a single amino acid, e.g. poly(lysine). Similarly,saccharides can be of mixed sequence or composed of a single saccharidesubunit, e.g., dextran, amylose, chitosan, and poly(sialic acid). Anexemplary poly(ether) is poly(ethylene glycol). Poly(ethylene imine) isan exemplary polyamine, and poly(aspartic) acid is a representativepoly(carboxylic acid).

“Poly(alkylene oxide)” refers to a genus of compounds having a polyetherbackbone. Poly(alkylene oxide) species of use in the present inventioninclude, for example, straight- and branched-chain species. Moreover,exemplary poly(alkylene oxide) species can terminate in one or morereactive, activatable, or inert groups. For example, poly(ethyleneglycol) is a poly(alkylene oxide) consisting of repeating ethylene oxidesubunits, which may or may not include additional reactive, activatableor inert moieties at either terminus. Useful poly(alkylene oxide)species include those in which one terminus is “capped” by an inertgroup, e.g., monomethoxy-poly(alkylene oxide). When the molecule is abranched species, it may include multiple reactive, activatable or inertgroups at the termini of the alkylene oxide chains and the reactivegroups may be either the same or different. Derivatives ofstraight-chain poly(alkylene oxide) species that are heterobifunctionalare also known in the art.

The term, “glycosyl linking group,” as used herein refers to a glycosylresidue to which an agent (e.g., water-soluble polymer, therapeuticmoiety, biomolecule) is covalently attached. In the methods of theinvention, the “glycosyl linking group” becomes covalently attached to aglycosylated or unglycosylated peptide, thereby linking the agent to anamino acid and/or glycosyl residue on the peptide. A “glycosyl linkinggroup” is generally derived from a “modified sugar” by the enzymaticattachment of the “modified sugar” to an amino acid and/or glycosylresidue of the peptide. More specifically, a “glycosyl linking group,”as used herein, refers to a moiety that covalently joins a “modifyinggroup,” as discussed herein, and an amino acid residue of a peptide. Theglycosyl linking group-modifying group adduct has a structure that is asubstrate for an enzyme. The enzymes for which the glycosyl linkinggroup-modifying group adduct are substrates are generally those capableof transferring a saccharyl moiety onto an amino acid residue of apeptide, e.g, a glycosyltransferase, amidase, glycosidase,trans-sialidase, etc. The “glycosyl linking group” is interposedbetween, and covalently joins a “modifying group” and an amino acidresidue of a peptide.

An “intact glycosyl linking group” refers to a linking group that isderived from a glycosyl moiety in which the individual saccharidemonomer that links the conjugate is not degraded, e.g., oxidized, e.g.,by sodium metaperiodate. “Intact glycosyl linking groups” of theinvention may be derived from a naturally occurring oligosaccharide byaddition of glycosyl unit(s) or removal of one or more glycosyl unitfrom a parent saccharide structure. An exemplary “intact glycosyllinking group” includes at least one intact, e.g., non-degraded,saccharyl moiety that is covalently attached to an amino acid residue ona peptide. The remainder of the “linking group” can have substantiallyany structure. For example, the modifying group is optionally linkeddirectly to the intact saccharyl moiety. Alternatively, the modifyinggroup is linked to the intact saccharyl moiety via a linker arm. Thelinker arm can have substantially any structure determined to be usefulin the selected embodiment. In an exemplary embodiment, the linker armis one or more intact saccharyl moieties, i.e. “the intact glycosyllinking group” resembles an oligosaccharide. Another exemplary intactglycosyl linking group is one in which a saccharyl moiety attached,directly or indirectly, to the intact saccharyl moiety is degraded andderivatized (e.g., periodate oxidation followed by reductive amination).Still a further linker arm includes the modifying group attached to theintact saccharyl moiety, directly or indirectly, via a cross-linker,such as those described herein or analogues thereof.

“Degradation,” as used herein refers to the removal of one or morecarbon atoms from a saccharyl moiety.

The terms “targeting moiety” and “targeting agent”, as used herein,refer to species that will selectively localize in a particular tissueor region of the body. The localization is mediated by specificrecognition of molecular determinants, molecular size of the targetingagent or conjugate, ionic interactions, hydrophobic interactions and thelike. Other mechanisms of targeting an agent to a particular tissue orregion are known to those of skill in the art.

As used herein, “therapeutic moiety” means any agent useful for therapyincluding, but not limited to, antibiotics, anti-inflammatory agents,anti-tumor drugs, cytotoxins, and radioactive agents. “Therapeuticmoiety” includes prodrugs of bioactive agents, constructs in which morethan one therapeutic moiety is linked to a carrier, e.g., multivalentagents. Therapeutic moiety also includes peptides, and constructs thatinclude peptides. Exemplary peptides include those disclosed in FIG. 28and Tables 6 and 7, herein. “Therapeutic moiety” thus means any agentuseful for therapy including, but not limited to, antibiotics,anti-inflammatory agents, anti-tumor drugs, cytotoxins, and radioactiveagents. “Therapeutic moiety” includes prodrugs of bioactive agents,constructs in which more than one therapeutic moiety is linked to acarrier, e.g., multivalent agents.

As used herein, “anti-tumor drug” means any agent useful to combatcancer including, but not limited to, cytotoxins and agents such asantimetabolites, alkylating agents, anthracyclines, antibiotics,antimitotic agents, procarbazine, hydroxyurea, asparaginase,corticosteroids, interferons and radioactive agents. Also encompassedwithin the scope of the term “anti-tumor drug,” are conjugates ofpeptides with anti-tumor activity, e.g. TNF-α. Conjugates include, butare not limited to those formed between a therapeutic protein and aglycoprotein of the invention. A representative conjugate is that formedbetween PSGL-1 and TNF-α.

As used herein, “a cytotoxin or cytotoxic agent” means any agent that isdetrimental to cells. Examples include taxol, cytochalasin B, gramicidinD, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracinedione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Other toxinsinclude, for example, ricin, CC-1065 and analogues, the duocarmycins.Still other toxins include diphtheria toxin, and snake venom (e.g.,cobra venom).

As used herein, “a radioactive agent” includes any radioisotope that iseffective in diagnosing or destroying a tumor. Examples include, but arenot limited to, indium-111, cobalt-60 and technetium. Additionally,naturally occurring radioactive elements such as uranium, radium, andthorium, which typically represent mixtures of radioisotopes, aresuitable examples of a radioactive agent. The metal ions are typicallychelated with an organic chelating moiety.

Many useful chelating groups, crown ethers, cryptands and the like areknown in the art and can be incorporated into the compounds of theinvention (e.g. EDTA, DTPA, DOTA, NTA, HDTA, etc. and their phosphonateanalogs such as DTPP, EDTP, HDTP, NTP, etc). See, for example, Pitt etal., “The Design of Chelating Agents for the Treatment of IronOverload,” In, INORGANIC CHEMISTRY IN BIOLOGY AND MEDICINE; Martell,Ed.; American Chemical Society, Washington, D.C., 1980, pp. 279-312;Lindoy, THE CHEMISTRY OF MACROCYCLIC LIGAND COMPLEXES; CambridgeUniversity Press, Cambridge, 1989; Dugas, BIOORGANIC CHEMISTRY;Springer-Verlag, New York, 1989, and references contained therein.

Additionally, a manifold of routes allowing the attachment of chelatingagents, crown ethers and cyclodextrins to other molecules is availableto those of skill in the art. See, for example, Meares et al.,“Properties of In Vivo Chelate-Tagged Proteins and Polypeptides.” In,MODIFICATION OF PROTEINS: FOOD, NUTRITIONAL, AND PHARMACOLOGICALASPECTS;” Feeney, et al., Eds., American Chemical Society, Washington,D.C., 1982, pp. 370-387; Kasina et al, Bioconjugate Chem., 9: 108-117(1998); Song et al, Bioconjugate Chlein., 8: 249-255 (1997).

As used herein, “pharmaceutically acceptable carrier” includes anymaterial, which when combined with the conjugate retains the activity ofthe conjugate activity and is non-reactive with the subject's immunesystem. Examples include, but are not limited to, any of the standardpharmaceutical carriers such as a phosphate buffered saline solution,water, emulsions such as oil/water emulsion, and various types ofwetting agents. Other carriers may also include sterile solutions,tablets including coated tablets and capsules. Typically such carrierscontain excipients such as starch, milk, sugar, certain types of clay,gelatin, stearic acid or salts thereof, magnesium or calcium stearate,talc, vegetable fats or oils, gums, glycols, or other known excipients.Such carriers may also include flavor and color additives or otheringredients. Compositions comprising such carriers are formulated bywell known conventional methods.

As used herein, “administering” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular, intralesional, intranasal orsubcutaneous administration, intrathecal administration, or theimplantation of a slow-release device e.g., a mini-osmotic pump, to thesubject.

The term “isolated” refers to a material that is substantially oressentially free from components, which are used to produce thematerial. For peptide conjugates of the invention, the term “isolated”refers to material that is substantially or essentially free fromcomponents, which normally accompany the material in the mixture used toprepare the peptide conjugate. “Isolated” and “pure” are usedinterchangeably. Typically, isolated peptide conjugates of the inventionhave a level of purity preferably expressed as a range. The lower end ofthe range of purity for the peptide conjugates is about 60%, about 70%or about 80% and the upper end of the range of purity is about 70%,about 80%, about 90% or more than about 90%.

When the peptide conjugates are more than about 90% pure, their puritiesare also preferably expressed as a range. The lower end of the range ofpurity is about 90%, about 92%, about 94%, about 96% or about 98%. Theupper end of the range of purity is about 92%, about 94%, about 96%,about 98% or about 100% purity.

Purity is determined by any art-recognized method of analysis (e.g.,band intensity on a silver stained gel, polyacrylamide gelelectrophoresis, HPLC, or a similar means).

“Commercial scale” as used herein means about one or more gram of finalproduct produced in the method.

“Essentially each member of the population,” as used herein, describes acharacteristic of a population of peptide conjugates of the invention inwhich a selected percentage of the modified sugars added to a peptideare added to multiple, identical acceptor sites on the peptide.“Essentially each member of the population” speaks to the “homogeneity”of the sites on the peptide conjugated to a modified sugar and refers toconjugates of the invention, which are at least about 80%, preferably atleast about 90% and more preferably at least about 95% homogenous.

“Homogeneity,” refers to the structural consistency across a populationof acceptor moieties to which the modified sugars are conjugated. Thus,in a peptide conjugate of the invention in which each modified sugar.moiety is conjugated to an acceptor site having the same structure asthe acceptor site to which every other modified sugar is conjugated, thepeptide conjugate is said to be about 100% homogeneous. Homogeneity istypically expressed as a range. The lower end of the range ofhomogeneity for the peptide conjugates is about 60%, about 70% or about80% and the upper end of the range of purity is about 70%, about 80%,about 90% or more than about 90%.

When the peptide conjugates are more than or equal to about 90%homogeneous, their homogeneity is also preferably expressed as a range.The lower end of the range of homogeneity is about 90%, about 92%, about94%, about 96% or about 98%. The upper end of the range of purity isabout 92%, about 94%, about 96%, about 98% or about 100% homogeneity.The purity of the peptide conjugates is typically determined by one ormore methods known to those of skill in the art, e.g., liquidchromatography-mass spectrometry (LC-MS), matrix assisted laserdesorption time of flight mass spectrometry (MALDI-TOF), capillaryelectrophoresis, and the like.

“Substantially uniform glycoform” or a “substantially uniformglycosylation pattern,” when referring to a glycopeptide species, refersto the percentage of acceptor moieties that are glycosylated by theglycosyltransferase of interest (e.g., fucosyltransferase). For example,in the case of a α1,2 fucosyltransferase, a substantially uniformfucosylation pattern exists if substantially all (as defined below) ofthe Galβ1,4-GlcNAc-R and sialylated analogues thereof are fucosylated ina peptide conjugate of the invention. It will be understood by one ofskill in the art, that the starting material may contain glycosylatedacceptor moieties (e.g., fucosylated Galβ1,4-GlcNAc-R moieties). Thus,the calculated percent glycosylation will include acceptor moieties thatare glycosylated by the methods of the invention, as well as thoseacceptor moieties already glycosylated in the starting material.

The term “substantially” in the above definitions of “substantiallyuniform” generally means at least about 40%, at least about 70%, atleast about 80%, or more preferably at least about 90%, and still morepreferably at least about 95% of the acceptor moieties for a particularglycosyltransferase are glycosylated.

Description of the Invention

I. Method to Remodel Glycan Chains

The present invention includes methods and compositions for the in vitroaddition and/or deletion of sugars to or from a glycopeptide molecule insuch a manner as to provide a peptide molecule having a specificcustomized or desired glycosylation pattern, preferably including theaddition of a modified sugar thereto. A key feature of the inventiontherefore is to take a peptide produced by any cell type and generate acore glycan structure on the peptide, following which the glycanstructure is then remodeled in vitro to generate a peptide having aglycosylation pattern suitable for therapeutic use in a mammal.

The importance of the glycosylation pattern of a peptide is well knownin the art as are the limitations of present in vivo methods for theproduction of properly glycosylated peptides, particularly when thesepeptides are produced using recombinant DNA methodology. Moreover, untilthe present invention, it has not been possible to generateglycopeptides having a desired glycan structure thereon, wherein thepeptide can be produced at industrial scale.

In the present invention, a peptide produced by a cell is enzymaticallytreated in vitro by the systematic addition of the appropriate enzymesand substrates therefor, such that sugar moieties that should not bepresent on the peptide are removed, and sugar moieties, optionallyincluding modified sugars, that should be added to the peptide are addedin a manner to provide a glycopeptide having “desired glycosylation”, asdefined elsewhere herein.

A. Method to Remodel N-linked Glycans

In one aspect, the present invention takes advantage of the fact thatmost peptides of commercial or pharmaceutical interest comprise a commonfive sugar structure referred to herein as the trimannosyl core, whichis N-linked to asparagine at the sequence Asn-X-Ser/Thr on a peptidechain. The elemental trimannosyl core consists essentially of twoN-acetylglucosamine (GlcNAc) residues and three mannose (Man) residuesattached to a peptide, i.e., it comprises these five sugar residues andno additional sugars, except that it may optionally include a fucoseresidue. The first GlcNAc is attached to the amide group of theasparagine and the second GlcNAc is attached to the first via a β1,4linkage. A mannose residue is attached to the second GlcNAc via a β1,4linkage and two mannose residues are attached to this mannose via anα1,3 and an α1,6 linkage respectively. A schematic depiction of atrimannosyl core structure is shown in FIG. 1, left side. While it isthe case that glycan structures on most peptides comprise other sugarsin addition to the trimannosyl core, the trimannosyl core structurerepresents an essential feature of N-linked glycans on mammalianpeptides.

The present invention includes the generation of a peptide having atrimannosyl core structure as a fundamental element of the structure ofthe glycan molecules contained thereon. Given the variety of cellularsystems used to produce peptides, whether the systems are themselvesnaturally occurring or whether they involve recombinant DNA methodology,the present invention provides methods whereby a glycan molecule on apeptide produced in any cell type can be reduced to an elementaltrimannosyl core structure. Once the elemental trimannosyl corestructure has been generated then it is possible using the methodsdescribed herein, to generate in vitro, a desired glycan structure onthe peptide which confers on the peptide one or more properties thatenhances the therapeutic effectiveness of the peptide.

It should be clear from the discussion herein that the term “trimannosylcore” is used to describe the glycan structure shown in FIG. 1, leftside. Glycopeptides having a trimannosyl core structure may also haveadditional sugars added thereto, and for the most part, do haveadditional structures added thereto irrespective of whether the sugarsgive rise to a peptide having a desired glycan structure. The term“elemental trimannosyl core structure” is defined elsewhere herein. Whenthe term “elemental” is not included in the description of the“trimannosyl core structure,” then the glycan comprises the trimannosylcore structure with additional sugars attached to the mannose sugars.

The term “elemental trimannosyl core glycopeptide” is used herein torefer to a glycopeptide having glycan structures comprised primarily ofan elemental trimannosyl core structure. However, it may also optionallycontain a fucose residue attached thereto. As discussed herein,elemental trimannosyl core glycopeptides are one optimal, and thereforepreferred, starting material for the glycan remodeling processes of theinvention.

Another optimal starting material for the glycan remodeling process ofthe invention is a glycan structure having a trimannosyl core whereinone or two additional GlcNAc residues are added to each of the α1,3 andthe α1,6 mannose residues (see for example, the structure on the secondline of FIG. 2, second structure in from the left of the figure). Thisstructure is referred to herein as “Man3GlcNAc4.” When the structure ismonoantennary, the structure is referred to herein as “Man3GlcNAc3.”Optionally, this structure may also contain a core fucose molecule. Oncethe Man3GlcNAc3 or Man3GlcNAc4 structure has been generated then it ispossible using the methods described herein, to generate in vitro, adesired glycan structure on the glycopeptide which confers on theglycopeptide one or more properties that enhances the therapeuticeffectiveness of the peptide.

In their native form, the N-linked glycopeptides of the invention, andparticularly the mammalian and human glycopeptides useful in the presentinvention, are N-linked glycosylated with a trimannosyl core structureand one or more sugars attached thereto.

The terms “glycopeptide” and “glycopolypeptide” are used synonymouslyherein to refer to peptide chains having sugar moieties attachedthereto. No distinction is made herein to differentiate smallglycopolypeptides or glycopeptides from large glycopolypeptides orglycopeptides. Thus, hormone molecules having very few amino acids intheir peptide chain (e.g., often as few as three amino acids) and othermuch larger peptides are included in the general terms“glycopolypeptide” and “glycopeptide,” provided they have sugar moietiesattached thereto. However, the use of the term “peptide” does notpreclude that peptide from being a glycopeptide.

An example of an N-linked glycopeptide having desired glycosylation is apeptide having an N-linked glycan having a trimannosyl core with atleast one GlcNAc residue attached thereto. This residue is added to thetrimannosyl core using N-acetyl glucosaminyltransferase I (GnT-I). If asecond GlcNAc residue is added, N-acetyl glucosaminyltransferase II(GnT-II) is used. Optionally, additional GlcNAc residues may be addedwith GnT-IV and/or GNT-V, and a third bisecting GlcNAc residue may beattached to the β1,4 mannose of the trimannosyl core using N-acetylglucosaminyltransferase III (GnT-III). Optionally, this structure may beextended by treatment with β1,4 galactosyltransferase to add a galactoseresidue to each non-bisecting GlcNAc, and even further optionally, usingα2,3 or α2,6-sialyltransferase enzymes, sialic acid residues may beadded to each galactose residue. The addition of a bisecting GlcNAc tothe glycan is not required for the subsequent addition of galactose andsialic acid residues; however, with respect to the substrate affinity ofthe rat and human GnT-III enzymes, the presence of one or more of thegalactose residues on the glycan precludes the addition of the bisectingGlcNAc in that the galactose-containing glycan is not a substrate forthese forms of GNT-III. Thus, in instances where the presence of thebisecting GlcNAc is desired and these forms of GnT-III are used, it isimportant should the glycan contain added galactose and/or sialicresidues, that they are removed prior to the addition of the bisectingGlcNAc. Other forms of GNT-III may not require this specific order ofsubstrates for their activity. In the more preferred reaction, a mixtureof GNT-I, GNT-II and GNT-III is added to the reaction mixture so thatthe GlcNAc residues can be added in any order.

Examples of glycan structures which represent the various aspects ofpeptides having “desired glycosylation” are shown in the drawingsprovided herein. The precise procedures for the in vitro generation of apeptide having “desired glycosylation” are described elsewhere herein.However, the invention should in no way be construed to be limitedsolely to any one glycan structure disclosed herein. Rather, theinvention should be construed to include any and all glycan structureswhich can be made using the methodology provided herein.

In some cases, an elemental trimannosyl core alone may constitute thedesired glycosylation of a peptide. For example, a peptide having only atrimannosyl core has been shown to be a useful component of an enzymeemployed to treat Gaucher disease (Mistry et al., 1966, Lancet 348:1555-1559; Bijsterbosch et al., 1996, Eur. J. Biochem. 237:344-349).

According to the present invention, the following procedures for thegeneration of peptides having desired glycosylation become apparent.

a) Beginning with a glycopeptide having one or more glycan moleculeswhich have as a common feature a trimannosyl core structure and at leastone or more of a heterogeneous or homogeneous mixture of one or moresugars added thereto, it is possible to increase the proportion ofglycopeptides having an elemental trimannosyl core structure as the soleglycan structure or which have Man3GlcNAc3 or Man3GlcNAc4 as the soleglycan structure. This is accomplished in vitro by the systematicaddition to the glycopeptide of an appropriate number of enzymes in anappropriate sequence which cleave the heterogeneous or homogeneousmixture of sugars on the glycan structure until it is reduced to anelemental trimannosyl core or Man3GlcNAc3 or Man3GlcNAc4 structure.Specific examples of how this is accomplished will depend on a varietyof factors including in large part the type of cell in which the peptideis produced and therefore the degree of complexity of the glycanstructure(s) present on the peptide initially produced by the cell.Examples of how a complex glycan structure can be reduced to anelemental trimannosyl core or a Man3GlcNAc3 or Man3GlcNAc4 structure arepresented in FIG. 2 or are described in detail elsewhere herein.

b) It is possible to generate a peptide having an elemental trimannosylcore structure as the sole glycan structure on the peptide by isolatinga naturally occurring cell whose glycosylation machinery produces such apeptide. DNA encoding a peptide of choice is then transfected into thecell wherein the DNA is transcribed, translated and glycosylated suchthat the peptide of choice has an elemental trimannosyl core structureas the sole glycan structure thereon. For example, a cell lacking afunctional GnT-I enzyme will produce several types of glycopeptides. Insome instances, these will be glycopeptides having no additional sugarsattached to the trimannosyl core. However, in other instances, thepeptides produced may have two additional mannose residues attached tothe trimannosyl core, resulting in a Man5 glycan. This is also a desiredstarting material for the remodeling process of the present invention.Specific examples of the generation of such glycan structures aredescribed herein.

c) Alternatively, it is possible to genetically engineer a cell toconfer upon it a specific glycosylation machinery such that a peptidehaving an elemental trimannosyl core or Man3GlcNAc3 or Man3GlcNAc4structure as the sole glycan structure on the peptide is produced. DNAencoding a peptide of choice is then transfected into the cell whereinthe DNA is transcribed, translated and glycosylated such that thepeptide of choice has an increased number of glycans comprising solelyan elemental trimannosyl core structure. For example, certain types ofcells that are genetically engineered to lack GnT-I, may produce aglycan having an elemental trimannosyl core structure, or, depending onthe cell, may produce a glycan having a trimannosyl core plus twoadditional mannose residues attached thereto (Man5). When the cellproduces a Man5 glycan structure, the cell may be further geneticallyengineered to express mannosidase 3 which cleaves off the two additionalmannose residues to generate the trimannosyl core. Alternatively, theMan5 glycan may be incubated in vitro with mannosidase 3 to have thesame effect.

d) When a peptide is expressed in an insect cell, the glycan on thepeptide comprises a partially complex chain. Insect cells also expresshexosaminidase in the cells which trims the partially complex chain backto a trimannosyl core structure which can then be remodeled as describedherein.

e) It is readily apparent from the discussion in b), c) and d) that itis not necessary that the cells produce only peptides having elementaltrimannosyl core or Man3GlcNAc3 or Man3GlcNAc4 structures attachedthereto. Rather, unless the cells described in b) and c) producepeptides having 100% elemental trimannosyl core structures (i.e., havingno additional sugars attached thereto) or 100% of Man3GlcNAc3 orMan3GlcNAc4 structures, the cells in fact produce a heterogeneousmixture of peptides having, in combination, elemental trimannosyl corestructures, or Man3GlcNAc3 or Man3GlcNAc4 structures, as the sole glycanstructure in addition to these structures having additional sugarsattached thereto. The proportion of peptides having a trimannosyl coreor Man3GlcNAc3 or Man3GlcNAc4 structures having additional sugarsattached thereto, as opposed to those having one structure, will varydepending on the cell which produces them. The complexity of the glycans(i.e. which and how many sugars are attached to the trimannosyl core)will also vary depending on the cell which produces them.

f) Once a glycopeptide having an elemental trimannosyl core or atrimannosyl core with one or two GlcNAc residues attached thereto isproduced by following a), b) or c) above, according to the presentinvention, additional sugar molecules are added in vitro to thetrimannosyl core structure to generate a peptide having desiredglycosylation (i.e., a peptide having an in vitro customized glycanstructure).

g) However, when it is the case that a peptide having an elementaltrimannosyl core or Man3GlcNAc4 structure with some but not all of thedesired sugars attached thereto is produced, then it is only necessaryto add any remaining desired sugars without reducing the glycanstructure to the elemental trimannosyl core or Man3GlcNAc4 structure.Therefore, in some cases, a peptide having a glycan structure having atrimannosyl core structure with additional sugars attached thereto, willbe a suitable substrate for remodeling.

Isolation of an Elemental Trimannosyl Core Glycopeptide

The elemental trimannosyl core or Man3GlcNAc3 or Man3GlcNAc4glycopeptides of the invention may be isolated and purified, ifnecessary, using techniques well known in the art of peptidepurification. Suitable techniques include chromatographic techniques,isoelectric focusing techniques, ultrafiltration techniques and thelike. Using any such techniques, a composition of the invention can beprepared in which the glycopeptides of the invention are isolated fromother peptides and from other components normally found within cellculture media. The degree of purification can be, for example, 90% withrespect to other peptides or 95%, or even higher, e.g., 98%. See, e.g.,Deutscher et al. (ed., 1990, Guide to Protein Purification, HarcourtBrace Jovanovich, San Diego).

The heterogeneity of N-linked glycans present in the glycopeptidesproduced by the prior art methodology generally only permits theisolation of a small portion of the target glycopeptides which can bemodified to produce desired glycopeptides. In the present methods, largequantities of elemental trimannosyl core glycopeptides and other desiredglycopeptides, including Man3GlcNAc3 or Man3GlcNAc4 glycans, can beproduced which can then be further modified to generate large quantitiesof peptides having desired glycosylation.

Specific enrichment of any particular type of glycan linked to a peptidemay be accomplished using lectins which have an affinity for the desiredglycan. Such techniques are well known in the art of glycobiology.

A key feature of the invention which is described in more detail below,is that once a core glycan structure is generated on any peptide, theglycan structure is then remodeled in vitro to generate a peptide havingdesired glycosylation that has improved therapeutic use in a mammal. Themammal may be any type of suitable mammal, and is preferably a human.

The various scenarios and the precise methods and compositions forgenerating peptides with desired glycosylation will become evident fromthe disclosure which follows.

The ultimate objective of the production of peptides for therapeutic usein mammals is that the peptides should comprise glycan structures thatfacilitate rather than negate the therapeutic benefit of the peptide. Asdisclosed throughout the present specification, peptides produced incells may be treated in vitro with a variety of enzymes which catalyzethe cleavage of sugars that should not be present on the glycan and theaddition of sugars which should be present on the glycan such that apeptide having desired glycosylation and thus suitable for therapeuticuse in mammals is generated. The generation of different glycoforms ofpeptides in cells is described above. A variety of mechanisms for thegeneration of peptides having desired glycosylation is now described,where the starting material i.e., the peptide produced by a cell maydiffer from one cell type to another. As will become apparent from thepresent disclosure, it is not necessary that the starting material beuniform with respect to its glycan composition. However, it ispreferable that the starting material be enriched for certain glycoformsin order that large quantities of end product, i.e., correctlyglycosylated peptides are produced.

In a preferred embodiment according to the present invention, thedegradation and synthesis events that result in a peptide having desiredglycosylation involve at some point, the generation of an elementaltrimannosyl core structure or a Man3GlcNAc3 or Man3GlcNAc4 structure onthe peptide.

The present invention also provides means of adding one or more selectedglycosyl residues to a peptide, after which a modified sugar isconjugated to at least one of the selected glycosyl residues of thepeptide. The present embodiment is useful, for example, when it isdesired to conjugate the modified sugar to a selected glycosyl residuethat is either not present on a peptide or is not present in a desiredamount. Thus, prior to coupling a modified sugar to a peptide, theselected glycosyl residue is conjugated to the peptide by enzymatic orchemical coupling. In another embodiment, the glycosylation pattern of apeptide is altered prior to the conjugation of the modified sugar by theremoval of a carbohydrate residue from the peptide. See for example WO98/31826.

Addition or removal of any carbohydrate moieties present on the peptideis accomplished either chemically or enzymatically. Chemicaldeglycosylation is preferably brought about by exposure of the peptidevariant to the compound trifluoromethanesulfonic acid, or an equivalentcompound. This treatment results in the cleavage of most or all sugarsexcept the linking sugar (N-acetylglucosamine or N-acetylgalactosamine),while leaving the peptide intact. Chemical deglycosylation is describedby Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259: 52 and by Edgeet al., 1981, Anal. Biochem. 118: 131. Enzymatic cleavage ofcarbohydrate moieties on peptide variants can be achieved by the use ofa variety of endo- and exo-glycosidases as described by Thotakura etal., 1987, Meth. Enzymol. 138: 350.

Chemical addition of glycosyl moieties is carried out by anyart-recognized method. Enzymatic addition of sugar moieties ispreferably achieved using a modification of the methods set forthherein, substituting native glycosyl units for the modified sugars usedin the invention. Other methods of adding sugar moieties are disclosedin U.S. Pat. Nos. 5,876,980, 6,030,815, 5,728,554, and 5,922,577.

Exemplary attachment points for selected glycosyl residue include, butare not limited to: (a) sites for N- and O-glycosylation; (b) terminalglycosyl moieties that are acceptors for a glycosyltransferase; (c)arginine, asparagine and histidine; (d) free carboxyl groups; (e) freesulfhydryl groups such as those of cysteine; (f) free hydroxyl groupssuch as those of serine, threonine, or hydroxyproline; (g) aromaticresidues such as those of phenylalanine, tyrosine, or tryptophan; or (h)the amide group of glutamine. Exemplary methods of use in the presentinvention are described in WO 87/05330 published Sep. 11, 1987, and inAplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Dealing specifically with the examples shown in several of the figuresprovided herein, a description of the sequence of in vitro enzymaticreactions for the production of desired glycan structures on peptides isnow presented. The precise reaction conditions for each of the enzymaticconversions disclosed below are well known to those skilled in the artof glycobiology and are therefore not repeated here. For a review of thereaction conditions for these types of reactions, see Sadler et al.,1982, Methods in Enzymology 83:458-514 and references cited therein.

In FIG. 1 there is shown the structure of an elemental trimannosyl coreglycan on the left side. It is possible to convert this structure to acomplete glycan structure having a bisecting GlcNAc by incubating theelemental trimannosyl core structure in the presence of GnT-I, followedby GnT-II, and further followed by GnT-III, and a sugar donor comprisingUDP-GlcNAc, wherein GlcNAc is sequentially added to the elementaltrimannosyl core structure to generate a trimannosyl core having abisecting GlcNAc. In some instances, for example when remodeling Fcglycans as described herein, the order of addition of GnT-I, GnT-II andGnT-III may be contrary to that reported in the literature. Thebisecting GlcNAc structure may be produced by adding a mixture of GnT-I,GnT-II and GnT-III and UDP-GlcNAc to the reaction mixture

In FIG. 3 there is shown the conversion of a bisecting GlcNAc containingtrimannosyl core glycan to a complex glycan structure comprisinggalactose and N-acetyl neuraminic acid. The bisecting GlcNAc containingtrimannosyl core glycan is first incubated with galactosyltransferaseand UDP-Gal as a donor molecule, wherein two galactose residues areadded to the peripheral GlcNAc residues on the molecule. The enzymeNeuAc-transferase is then used to add two NeuAc residues one to each ofthe galactose residues.

In FIG. 4 there is shown the conversion of a high mannose glycanstructure to an elemental trimannosyl core glycan. The high mannoseglycan (Man9) is incubated sequentially in the presence of themannosidase 1 to generate a Man5 structure and then in the presence ofmannosidase 3, wherein all but three mannose residues are removed fromthe glycan. Alternatively, incubation of the Man9 structure may betrimmed back to the trimannosyl core structure solely by incubation inthe presence of mannosidase 3. According to the schemes presented inFIGS. 1 and 3 above, conversion of this elemental trimannosyl coreglycan to a complex glycan molecule is then possible.

In FIG. 5 there is shown a typical complex N-linked glycan structureproduced in plant cells. It is important to note that when plant cellsare deficient in GnT-I enzymatic activity, xylose and fucose cannot beadded to the glycan. Thus, the use of GnT-I knock-out cells provides aparticular advantage in the present invention in that these cellsproduce peptides having an elemental trimannosyl core onto whichadditional sugars can be added without performing any “trimming back”reactions. Similarly, in instances where the structure produced in aplant cell may be of the Man5 variety of glycan, if GnT-I is absent inthese cells, xylose and fucose cannot be added to the structure. In thiscase, the Man5 structure may be trimmed-back to an elemental trimannosylcore (Man3) using mannosidase 3. According to the methods providedherein, it is now possible to add desired sugar moieties to thetrimannosyl core to generate a desired glycan structure.

In FIG. 6 there is shown a typical complex N-linked glycan structureproduced in insect cells. As is evident, additional sugars, such as, forexample, fucose may also be present. Further although not shown here,insect cells may produce high mannose glycans having as many as ninemannose residues and may have additional sugars attached thereto. It isalso the case in insect cells that GnT-I knock out cells prevent theaddition of fucose residues to the glycan. Thus, production of a peptidein insect cells may preferably be accomplished in a GnT-I knock outcell. The glycan thus produced may then be trimmed back in vitro ifnecessary using any of the methods and schemes described herein, andadditional sugars may be added in vitro thereto also using the methodsand schemes provided herein.

In FIG. 2 there is shown glycan structures in various stages ofcompletion. Specifically, the in vitro enzymatic generation of anelemental trimannosyl core structure from a complex carbohydrate glycanstructure which does not contain a bisecting GlcNAc residue is shown.Also shown is the generation of a glycan structure therefrom whichcontains a bisecting GlcNAc. Several intermediate glycan structureswhich can be produced are shown. These structures can be produced bycells, or can be produced in the in vitro trimming back reactionsdescribed herein. Sugar moieties may be added in vitro to the elementaltrimannosyl core structure, or to any suitable intermediate structure inorder that a desired glycan is produced.

In FIG. 7 there is shown a series of possible in vitro reactions whichcan be performed to trim back and add onto glycans beginning with a highmannose structure. For example, a Man9 glycan may be trimmed usingmannosidase 1 to generate a Man5 glycan, or it may be trimmed to atrimannosyl core using mannosidase 3 or one or more microbialmannosidases. GnT-I and or GnT-II may then be used to transferadditional GlcNAc residues onto the glycan. Further, there is shown thesituation which would not occur when the glycan molecule is produced ina cell that does not have GnT-I (see shaded box). For example, fucoseand xylose may be added to a glycan only when GnT-I is active andfacilitates the transfer of a GlcNAc to the molecule.

FIG. 8 depicts well known strategies for the synthesis of biantennary,triantennary and even tetraantennary glycan structures beginning withthe trimannosyl core structure. According to the methods of theinvention, it is possible to synthesize each of these structures invitro using the appropriate enzymes and reaction conditions well knownin the art of glycobiology.

FIG. 9 depicts two methods for synthesis of a monoantennary glycanstructure beginning from a high mannose (6 to 9 mannose moieties) glycanstructures. A terminal sialic acid-PEG moiety may be added in place ofthe sialic acid moiety in accordance with glycoPEGylation methodologydescribed herein. In the first method, endo-H is used to cleave theglycan structure on the peptide back to the first GlcNAc residue.Galactose is then added using galactosyltransferase and sialylated-PEGis added as described elsewhere herein. In the second method,mannosidase I is used to cleave mannose residues from the glycanstructure in the peptide. A galactose residue is added to one arm of theremaining mannose residues which were cleaved off the glycan using JackBean α-mannosidase. Sialylated-PEG is then added to this structure asdirected.

FIG. 10 depicts two additional methods for synthesis of a monoantennaryglycan structures beginning from high mannose (6 to 9 mannose moieties)glycan structure. As in FIG. 9, a terminal sialic acid-PEG moiety may beadded in place of the sialic acid moiety in accordance with theglycoPEGylation methodology described herein. In the situation describedhere, some of the mannose residues from the arm to which sialylated-PEGis not added, are removed.

In FIG. 11 there is shown a scheme for the synthesis of yet more complexcarbohydrate structures beginning with a trimannosyl core structure. Forexample, a scheme for the in vitro production of Lewis x and Lewis aantigen structures, which may or may not be sialylated is shown. Suchstructures when present on a peptide may confer on the peptideimmunological advantages for upregulating or downregulating the immuneresponse. In addition, such structures are useful for targeting thepeptide to specific cells, in that these types of structures areinvolved in binding to cell adhesion peptides and the like.

FIG. 12 is an exemplary scheme for preparing an array of O-linkedpeptides originating with serine or threonine.

FIG. 13 is a series of diagrams depicting the four types of O-linkedglycan structure termed cores 1 through 4. The core structure isoutlined in dotted lines. Sugars which may also be included in thisstructure include sialic acid residues added to the galactose residues,and fucose residues added to the GlcNAc residues.

Thus, in preferred embodiments, the present invention provides a methodof making an N-linked glycosylated glycopeptide by providing an isolatedand purified glycopeptide to which is attached an elemental trimannosylcore or a Man3GlcNAc4 structure, contacting the glycopeptide with aglycosyltransferase enzyme and a donor molecule having a glycosyl moietyunder conditions suitable to transfer the glycosyl moiety to theglycopeptide. Customization of a trimannosyl core glycopeptide orMan3GlcNAc4 glycopeptide to produce a peptide having a desiredglycosylation pattern is then accomplished by the sequential addition ofthe desired sugar moieties, using techniques well known in the art.

Determination of Glycan Primary Structure

When an N-linked glycopeptide is produced by a cell, as noted elsewhereherein, it may comprise a heterogeneous mixture of glycan structureswhich must be reduced to a common, generally elemental trimannosyl coreor Man3GlcNAc4 structure, prior to adding other sugar moieties thereto.In order to determine exactly which sugars should be removed from anyparticular glycan structure, it is sometimes necessary that the primaryglycan structure be identified. Techniques for the determination ofglycan primary structure are well know in the art and are described indetail, for example, in Montreuil, “Structure and Biosynthesis ofGlycopeptides” In Polysaccharides in Medicinal Applications, pp.273-327, 1996, Eds. Severian Damitriu, Marcel Dekker, NY. It istherefore a simple matter for one skilled in the art of glycobiology toisolate a population of peptides produced by a cell and determine thestructure(s) of the glycans attached thereto. For example, efficientmethods are available for (i) the splitting of glycosidic bonds eitherby chemical cleavage such as hydrolysis, acetolysis, hydrazinolysis, orby nitrous deamination; (ii) complete methylation followed by hydrolysisor methanolysis and by gas-liquid chromatography and mass spectroscopyof the partially methylated monosaccharides; and (iii) the definition ofanomeric linkages between monosaccharides using exoglycosidases, whichalso provide insight into the primary glycan structure by sequentialdegradation. In particular, the techniques of mass spectroscopy andnuclear magnetic resonance (NMR) spectrometry, especially high field NMRhave been successfully used to determine glycan primary structure.

Kits and equipment for carbohydrate analysis are also commerciallyavailable. Fluorophore Assisted Carbohydrate Electrophoresis (FACE®) isavailable from Glyko, Inc. (Novato, Calif.). In FACE analysis,glycoconjugates are released from the peptide with either Endo H orN-glycanase (PNGase F) for N-linked glycans, or hydrazine for Ser/Thrlinked glycans. The glycan is then labeled at the reducing end with afluorophore in a non-structure discriminating manner. The fluorophorelabeled glycans are then separated in polyacrylamide gels based on thecharge/mass ratio of the saccharide as well as the hydrodynamic volume.Images are taken of the gel under UV light and the composition of theglycans are determined by the migration distance as compared with thestandards. Oligosaccharides can be sequenced in this manner by analyzingmigration shifts due to the sequential removal of saccharides byexoglycosidase digestion.

Exemplary Embodiment

The remodeling of N-linked glycosylation is best illustrated withreference to Formula 1:

where X³, X⁴, X⁵, X⁶, X⁷ and X¹⁷ are (independently selected)monosaccharide or oligosaccharide residues; and

a, b, c, d, e and x are (independently selected) 0, 1 or 2, with theproviso that at least one member selected from a, b, c, d, e and x are 1or 2.

Formula 1 describes glycan structure comprising the tri-mannosyl core,which is preferably covalently linked to an asparagine residue on apeptide backbone. Preferred expression systems will express and secreteexogenous peptides with N-linked glycans comprising the tri-mannosylcore. Using the remodeling method of the invention, the glycanstructures on these peptides can be conveniently remodeled to any glycanstructure desired. Exemplary reaction conditions are found throughoutthe examples and in the literature.

In preferred embodiments, the glycan structures are remodeled so thatthe structure described in Formula 1 has specific determinates. Thestructure of the glycan can be chosen to enhance the biological activityof the peptide, give the peptide a new biological activity, remove thebiological activity of peptide, or better approximate the glycosylationpattern of the native peptide, among others.

In the first preferred embodiment, the peptide N-linked glycans areremodeled to better approximate the glycosylation pattern of nativehuman proteins. In this embodiment, the glycan structure described inFormula 1 is remodeled to have the following moieties:

-   -   X³ and X⁵=|-GlcNAc-Gal-SA;    -   a and c=1;    -   d=0 or 1;    -   b, e and x=0.        This embodiment is particularly advantageous for human peptides        expressed in heterologous cellular expression systems. By        remodeling the N-linked glycan structures to this configuration,        the peptide can be made less immunogenic in a human patient,        and/or more stable, among others.

In the second preferred embodiment, the peptide N-linked glycans areremodeled to have a bisecting GlcNAc residue on the tri-mannosyl core.In this embodiment, the glycan structure described in Formula 1 isremodeled to have the following moieties:

-   -   X³ and X⁵ are |-GlcNAc-Gal-SA;    -   a and c=1;    -   X⁴ is GlcNAc;    -   b=1;    -   d=0 or 1;    -   e and x=0.        This embodiment is particularly advantageous for recombinant        antibody molecules expressed in heterologous cellular systems.        When the antibody molecule includes a Fc-mediated cellular        cytotoxicity, it is known that the presence of bisected        oligosaccharides linked the Fc domain dramatically increased        antibody-dependent cellular cytotoxicity.

In a third preferred embodiment, the peptide N-linked glycans areremodeled to have a sialylated Lewis X moiety. In this embodiment, theglycan structure described in Formula 1 is remodeled to have thefollowing moieties:

This embodiment is particularly advantageous when the peptide which isbeing remodeling is intended to be targeted to selectin molecules andcells exhibiting the same.

In a fourth preferred embodiment, the peptide N-linked glycans areremodeled to have a conjugated moiety. The conjugated moiety may be aPEG molecule, another peptide, a small molecule such as a drug, amongothers. In this embodiment, the glycan structure described in Formula 1is remodeled to have the following moieties:

-   -   X³ and X⁵ are |-GlcNAc-Gal-SA-R;    -   a and c=1 or 2;    -   d=0 or 1;    -   b, d, e and x=0;    -   where R=conjugate group.        The conjugated moiety may be a PEG molecule, another peptide, a        small molecule such as a drug, among others. This embodiment        therefore is useful for conjugating the peptide to PEG molecules        that will slow the clearance of the peptide from the patient's        bloodstream, to peptides that will target both peptides to a        specific tissue or cell, or to another peptide of complementary        therapeutic use.

It will be clear to one of skill in the art that the invention is notlimited to the preferred glycan molecules described above. The preferredembodiments are only a few of the many useful glycan molecules that canbe made by the remodeling method of the invention. Those skilled in theart will know how to design other useful glycans.

In the first exemplary embodiments, the peptide is expressed in a CHO(Chinese hamster ovarian cell line) according to methods well known inthe art. When a peptide with N-linked glycan consensus sites isexpressed and secreted from CHO cells, the N-linked glycans will havethe structures depicted in top row of FIG. 2, but also comprising a corefucose. While all of these structures may be present, by far the mostcommon structures are the two at the right side. In the terms of Formula1,

-   -   X³ and X⁵ are |-GlcNAc-Gal-(SA);    -   a and c=1;    -   b, e and x=0, and    -   d=0 or 1.        Therefore, in one exemplary embodiment, the N-linked glycans of        peptides expressed in CHO cells are remodeled to the preferred        humanized glycan by contacting the peptides with a        glycosyltransferase that is specific for a galactose acceptor        molecule and a sialic acid donor molecule. This process is        illustrated in FIG. 2 and Example 17. In another exemplary        embodiment, the N-linked glycans of a peptide expressed and        secreted from CHO cells are remodeled to be the preferred        PEGylated structures. The peptide is first contacted with a        glycosidase specific for sialic acid to remove the terminal SA        moiety, and then contacted with a glycosyltransferase specific        for a galactose acceptor moiety and an sialic acid acceptor        moiety, in the presence of PEG-sialic acid-nucleotide donor        molecules. Optionally, the peptide may then be contacted with a        glycosyltransferase specific for a galactose acceptor moiety and        an sialic acid acceptor moiety, in the presence of sialic        acid-nucleotide donor molecules to ensure complete the SA        capping of all of the glycan molecules.

In other exemplary embodiments, the peptide is expressed in insectcells, such as the sf9 cell line, according to methods well known in theart. When a peptide with N-linked glycan consensus sites is expressedand secreted from sf9 cells, the N-linked glycans will often have thestructures depicted in top row of FIG. 6. In the terms of Formula 1:

-   -   X³ and X⁵ are |-GlcNAc;    -   a and c=0 or 1;    -   b=0;    -   X⁶ is fucose,    -   d=0, 1 or 2; and    -   e and x=0.

The trimannose core is present in the vast majority of the N-linkedglycans made by insect cells, and sometimes an antennary GlcNAc and/orfucose residue(s) are also present. Note that the glycan may have nocore fucose, it may have a single core fucose having either linkage, orit may have a single core fucose with a perponderance of a singlelinkage. In one exemplary embodiment, the N-linked glycans of a peptideexpressed and secreted from insect cells is remodeled to the preferredhumanized glycan by first contacting the glycans with a glycosidasespecific to fucose molecules, then contacting the glycans with aglycosyltransferases specific to the mannose acceptor molecule on eachantennary of the trimannose core, a GlcNAc donor molecule in thepresence of nucleotide-GlcNAc molecules; then contacting the glycanswith a glycosyltransferase specific to a GlcNAc acceptor molecule, a Galdonor molecule in the presence of nucleotide-Gal molecules; and thencontacting the glycans with a glycosyltransferase specific to agalactose acceptor molecule, a sialic acid donor molecule in thepresence of nucleotide-SA molecules. One of skill in the art willappreciate that the fucose molecules, if any, can be removed at any timeduring the procedure, and if the core fucose is of the same alpha 1,6linkage as found in human glycans, it may be left intact. In anotherexemplary embodiment, the humanized glycan of the previous example isremodeled further to the sialylated Lewis X glycan by contacting theglycan further with a glycosyltransferase specific to a GlcNAc acceptormolecule, a fucose donor molecule in the presence of nucleotide-fucosemolecules. This process is illustrated in FIG. 11 and Example 39.

In yet other exemplary embodiments, the peptide is expressed in yeast,such as Saccharomyces cerevisiae, according to methods well known in theart. When a peptide with N-linked glycan consensus sites is expressedand secreted from S. cerevisiae cells, the N-linked glycans will havethe structures depicted at the left in FIG. 4. The N-inked glycans willalways have the trimannosyl core, which will often be elaborated withmannose or related polysaccharides of up to 1000 residues. In the termsof Formula 1:

-   -   X³ and X⁵=|-Man-Man-(Man)₀₋₁₀₀;    -   a and c=1 or 2;    -   b, d, e and x=0.

In one exemplary embodiment, the N-linked glycans of a peptide expressedand secreted from yeast cells are remodeled to the elemental trimannosecore by first contacting the glycans with a glycosidase specific to α2mannose molecules, then contacting the glycans with a glycosidasespecific to α6 mannose molecules. This process is illustrated in FIG. 4and Example 38.

In another exemplary embodiment, the N-linked glycans are furtherremodeled to make a glycan suitable for an recombinant antibody withFc-mediated cellular toxicity function by contacting the elementaltrimannose core glycans with a glycosyltransferase specific to themannose acceptor molecule on each antennary of the trimannose core and aGlcNAc donor molecule in the presence of nucleotide-GlcNAc molecules.Then, the glycans are contacted with a glycosyltransferase specific tothe acceptor mannose molecule in the middle of the trimannose core, aGlcNAc donor molecule in the presence of nucleotide-GlcNAc molecules andfurther contacting the glycans with a glycosyltransferase specific to aGlcNAc acceptor molecule, a Gal donor molecule in the presence ofnucleotide-Gal molecules; and then optionally contacting the glycanswith a glycosyltransferase specific to a galactose acceptor molecule andfurther optionally a sialic acid donor molecule in the presence ofnucleotide-SA molecules. This process is illustrated in FIGS. 1, 2 and3.

In another exemplary embodiment, the peptide is expressed in bacterialcells, in particular E. coli cells, according to methods well known inthe art. When a peptide with N-linked glycans consensus sites isexpressed in E. coli cells, the N-linked consensus sites will not beglycosylated. In an exemplary embodiment, a humanized glycan molecule isbuilt out from the peptide backbone by contacting the peptides with aglycosyltransferase specific for a N-linked consensus site and a GlcNAcdonor molecule in the presence of nucleotide-GlcNAc; and furthersequentially contacting the growing glycans with glycosyltransferasesspecific for the acceptor and donor moieties in the present of therequired donor moiety until the desired glycan structure is completed.When a peptide with N-linked glycans is expressed in a eukaryotic cellsbut without the proper leader sequences that direct the nascent peptideto the golgi apparatus, the mature peptide is likely not to beglycosylated. In this case as well the peptide may be given N-linkedglycosylation by building out from the peptide N-linked consensus siteas aforementioned. When a protein is chemically modified with a sugarmoiety, it can be built out as aforementioned.

These examples are meant to illustrate the invention, and not to limitit. One of skill in the art will appreciate that the steps taken in eachexample may in some circumstances be able to be performed in a differentorder to get the same result. One of skill in the art will alsounderstand that a different set of steps may also produce the sameresulting glycan. The preferred remodeled glycan is by no means specificto the expression system that the peptide is expressed in. The remodeledglycans are only illustrative and one of skill in the art will know howto take the principles from these examples and apply them to peptidesproduced in different expression systems to make glycans notspecifically described herein.

B. Method to Remodel O-linked Glycans

O-glycosylation is characterized by the attachment of a variety ofmonosaccharides in an O-glycosidic linkage to hydroxy amino acids.O-glycosylation is a widespread post-translational modification in theanimal and plant kingdoms. The structural complexity of glycans O-linkedto proteins vastly exceeds that of N-linked glycans. Serine or threonineresidues of a newly translated peptide become modified by virtue of apeptidyl GalNAc transferase in the cis to trans compartments of theGolgi. The site of O-glycosylation is determined not only by thesequence specificity of the glycosyltransferase, but also epigeneticregulation mediated by competition between different substrate sites andcompetition with other glycosyltransferases responsible for forming theglycan.

The O-linked glycan has been arbitrarily defined as having threeregions: the core, the backbone region and the peripheral region. The“core” region of an O-linked glycan is the inner most two or threesugars of the glycan chain proximal to the peptide. The backbone regionmainly contributes to the length of the glycan chain formed by uniformelongation. The peripheral region exhibits a high degree of structuralcomplexity. The structural complexity of the O-linked glycans beginswith the core structure. In most cases, the first sugar residue added atthe O-linked glycan consensus site is GalNAc; however the sugar may alsobe GlcNAc, glucose, mannose, galactose or fucose, among others. FIG. 12is a diagram of some of the known O-linked glycan core structures andthe enzymes responsible for their in vivo synthesis.

In mammalian cells, at least eight different O-linked core structuresare found, all based on a core-α-GalNAc residue. The four corestructures depicted in FIG. 13 are the most common. Core 1 and core 2are the most abundant structures in mammalian cells, and core 3 and core4 are found in more restricted, organ-characteristic expression systems.O-linked glycans are reviewed in Montreuil, Structure and Synthesis ofGlycopeptides, In Polysaccharides in Medicinal Applications, pp.273-327, 1996, Eds. Severian Damitriu, Marcel Dekker, NY, and inSchachter and Brockhausen, The Biosynthesis of Branched O-LinkedGlycans, 1989, Society for Experimental Biology, pp. 1-26 (GreatBritain).

It will be apparent from the present disclosure that the glycanstructure of O-glycosylated peptides can be remodeled using similartechniques to those described for N-linked glycans. O-glycans differfrom N-glycans in that they are linked to a serine or threonine residuerather than an asparagine residue. As described herein with respect toN-glycan remodeling, hydrolytic enzymes can be used to cleave unwantedsugar moieties in an O-linked glycan and additional desired sugars canthen be added thereto, to build a customized O-glycan structure on thepeptide (See FIGS. 12 and 13).

The initial step in O-glycosylation in mammalian cells is the attachmentof N-acetylgalactosamine (GalNAc) using any of a family of at leasteleven known α-N-acetylgalactosaminyltransferases, each of which has arestricted acceptor peptide specificity. Generally, the acceptor peptiderecognized by each enzyme constitutes a sequence of at least ten aminoacids. Peptides that contain the amino acid sequence recognized by oneparticular GalNAc-transferase become O-glycosylated at the acceptor siteif they are expressed in a cell expressing the enzyme and if they areappropriately localized to the Golgi apparatus where UDP-GalNAc is alsopresent.

However, in the case of recombinant proteins, the initial attachment ofthe GalNAc may not take place. The α-N-acetylgalactosaminyltransferaseenzyme native to the expressing cell may have a consensus sequencespecificity which differs from that of the recombinant peptide beingexpressed.

The desired recombinant peptide may be expressed in a bacterial cell,such as E. coli, that does not synthesize glycan chains. In these cases,it is advantageous to add the initial GalNAc moiety in vitro. The GalNAcmoiety can be introduced in vitro onto the peptide once the recombinantpeptide has been recovered in a soluble form, by contacting the peptidewith the appropriate GalNAc transferase in the presence of UDP-GalNAc.

In one embodiment, an additional sequence of amino acids that constitutean effective acceptor for transfer of an O-linked sugar may be present.Such an amino acid sequence is encoded by a DNA sequence fused in frameto the coding sequence of the peptide, or alternatively, may beintroduced by chemical means. The peptide may be otherwise lackingglycan chains. Alternately, the peptide may have N- and/or O-linkedglycan chains but require an additional glycosylation site, for example,when an additional glycan substituent is desired.

In an exemplary embodiment, the amino acid sequence PTTTK-COOH, which isthe natural GalNAc acceptor sequence in the human mucin MUC-1, is addedas a fusion tag. The fusion protein is then expressed in E. coli andpurified. The peptide is then contacted with recombinant humanGalNAc-transferases T3 or T6 in the presence of UDP-GalNAc to transfer aGalNAc residue onto the peptide in vitro.

This glycan chain on the peptide may then be further elongated using themethods described in reference to the N-linked or O-linked glycansherein. Alternatively, the GalNAc transferase reaction can be carriedout in the presence of UDP-GalNAc to which PEG is covalently substitutedin the O-3, 4, or 6 positions or the N-2 position. Glycoconjugation isdescribed in detail elswhere herein. Any antigenicity introduced intothe peptide by the new peptide sequence can be conveniently masked byPEGylation of the associated glycan. The acceptor site fusion techniquecan be used to introduce not only a PEG moiety, but to introduce otherglycan and non-glycan moieties, including, but not limited to, toxins,anti-infectives, cytotoxic agents, chelators for radionucleotides, andglycans with other functionalities, such as tissue targeting.

Exemplary Embodiments

The remodeling of O-linked glycosylation is best illustrated withreference to Formula 2:

Formula 2 describes a glycan structure comprising a GalNAc which iscovalently linked preferably to a serine or threonine residue on apeptide backbone. While this structure is used to illustrate the mostcommon forms of O-linked glycans, it should not be construed to limitthe invention solely to these O-linked glycans. Other forms of O-linkedglycans are illustrated in FIG. 12. Preferred expression systems usefulin the present invention express and secrete exogenous peptides havingO-linked glycans comprising the GalNAc residue. Using the remodelingmethods of the invention, the glycan structures on these peptides can beconveniently remodeled to generate any desired glycan structure. One ofskill in the art will appreciate that O-linked glycans can be remodeledusing the same principles, enzymes and reaction conditions as thoseavailable in the art once armed with the present disclosure. Exemplaryreaction conditions are found throughout the Examples.

In preferred embodiments, the glycan structures are remodeled so thatthe structure described in Formula 2 has specific moieties. Thestructure of the glycan may be chosen to enhance the biological activityof the peptide, confer upon the peptide a new biological activity,remove or alter a biological activity of peptide, or better approximatethe glycosylation pattern of the native peptide, among others.

In the first preferred embodiment, the peptide O-linked glycans areremodeled to better approximate the glycosylation pattern of nativehuman proteins. In this embodiment, the glycan structure described inFormula 2 is remodeled to have the following moieties:

-   -   X² is |-SA; or |-SA-SA;    -   f and n=0 or 1;    -   X¹⁰ is SA;    -   m=0.        This embodiment is particularly advantageous for human peptides        expressed in heterologous cellular expression systems. By        remodeling the O-linked glycan structures to have this        configuration, the peptide can be rendered less immunogenic in a        human patient and/or more stable.

In the another preferred embodiment, the peptide O-linked glycans areremodeled to display a sialylated Lewis X antigen. In this embodiment,the glycan structure described in Formula 2 is remodeled to have thefollowing moieties:

-   -   X² is |-SA;    -   X¹⁰ is Fuc or |-GlcNAc(Fuc)-Gal-SA;    -   f and n=1;    -   m=0.        This embodiment is particularly advantageous when the peptide        which is being remodeled is most effective when targeted to a        selectin molecule and cells exhibiting the same.

In a yet another preferred embodiment, the peptide O-linked glycans areremodeled to contain a conjugated moiety. The conjugated moiety may be aPEG molecule, another peptide, a small molecule such as a drug, amongothers. In this embodiment, the glycan structure described in Formula 2is remodeled to have the following moieties:

-   -   X² is |-SA-R;    -   f=1;    -   n and m=0;    -   where R is the conjugate group.        This embodiment is useful for conjugating the peptide to PEG        molecules that will slow the clearance of the peptide from the        patient's bloodstream, to peptides that will target both        peptides to a specific tissue or cell or to another peptide of        complementary therapeutic use.

It will be clear to one of skill in the art that the invention is notlimited to the preferred glycan molecules described above. The preferredembodiments are only a few of the many useful glycan molecules that canbe made using the remodeling methods of the invention. Those skilled inthe art will know how to design other useful glycans once armed with thepresent invention.

In the first exemplary embodiment, the peptide is expressed in a CHO(Chinese hamster cell line) according to methods well known in the art.When a peptide with O-linked glycan consensus sites is expressed andsecreted from CHO cells, the majority of the O-linked glycans will oftenhave the structure, in the terms of Formula 2,

-   -   X²=|-SA;    -   f=1;    -   m and n=0.        Therefore, most of the glycans in CHO cells do not require        remodeling in order to be acceptable for use in a human patient.        In an exemplary embodiment, the O-linked glycans of a peptide        expressed and secreted from a CHO cell are remodeled to contain        a sialylated Lewis X structure by contacting the glycans with a        glycosyltransferase specific for the GalNAc acceptor moiety and        the fucose donor moiety in the presence of nucleotide-fucose.        This process is illustrated on N-linked glycans in FIG. 11 and        Example 39.

In other exemplary embodiments, the peptide is expressed in insect cellssuch as sf9 according to methods well known in the art. When a peptidehaving O-inked glycan consensus sites is expressed and secreted frommost sf9 cells, the majority of the O-linked glycans have the structure,in the terms of Formula 2:

-   -   X²=H;    -   f=0 or 1;    -   n and m=0.        See, for example, Marchal et al., (2001, Biol. Chem.        382:151-159). In one exemplary embodiment, the O-linked glycan        on a peptide expressed in an insect cell is remodeled to a        humanized glycan by contacting the glycans with a        glycosyltransferase specific for a GalNAc acceptor molecule and        a galactose donor molecule in the presence of nucleotide-Gal;        and then contacting the glycans with a glycosyltransferase        specific for a Gal acceptor molecule and a SA donor molecule in        the presence of nucleotide-SA. In another exemplary embodiment,        the O-linked glycans are remodeled further from the humanized        form to the sialylated Lewis X form by further contacting the        glycans with a glycosyltransferase specific for a GalNAc        acceptor molecule and a fucose donor molecule in the presence of        nucleotide-fucose.

In yet another exemplary embodiment, the peptide is expressed in fungalcells, in particular S. cerevisiae cells, according to methods wellknown in the art. When a peptide with O-linked glycans consensus sitesis expressed and secreted from S. cerevisiae cells, the majority of theO-linked glycans have the structure:

|-AA-Man-Man₁₋₂.

See Gemmill and Trimble (1999, Biochim. Biophys. Acta 1426:227-237). Inorder to remodel these O-linked glycans for use in human, it ispreferable that the glycan be cleaved at the amino acid level andrebuilt from there.

In an exemplary embodiment, the glycan is the O-linked glycan on apeptide expressed in a fungal cell and is remodeled to a humanizedglycan by contacting the glycan with an endoglycosylase specific for anamino acid—GalNAc bond; and then contacting the glycan with aglycosyltransferase specific for a O-linked consensus site and a GalNAcdonor molecule in the presence of nucleotide-GalNAc; contacting theglycan with a glycosyltransferase specific for a GalNAc acceptormolecule and a galactose donor molecule in the presence ofnucleotide-Gal; and then contacting the glycans with aglycosyltransferase specific for a Gal acceptor molecule and a SA donormolecule in the presence of nucleotide-SA.

Alternately, in another exemplary embodiment, the glycan is the O-linkedglycan on a peptide expressed in a fungal cell and is remodeled to ahumanized glycan by contacting the glycan with an protein O-mannoseβ-1,2-N-acetylglucosaminyltransferase (POMGnTI) in the presence ofGlcNAc-nucleotide; then contacting the glycan with angalactosyltransferase in the presence of nucleotide-Gal; and thencontracting the glycan with an sialyltransferase in the presence ofnucleotide-SA.

In another exemplary embodiment, the peptide is expressed in bacterialcells, in particular E. coli cells, according to methods well known inthe art. When a peptide with an O-linked glycan consensus site isexpressed in E. coli cells, the O-linked consensus site will not beglycosylated. In this case, the desired glycan molecule must be builtout from the peptide backbone in a manner similar to that describe forS. cerevisiae expression above. Further, when a peptide having anO-linked glycan is expressed in a eukaryotic cell without the properleader sequences to direct the nascent peptide to the golgi apparatus,the mature peptide is likely not to be glycosylated. In this case aswell, an O-linked glycosyl structure may be added to the peptide bybuilding out the glycan directly from the peptide O-linked consensussite. Further, when a protein is chemically modified with a sugarmoiety, it can also be remodeled as described herein.

These examples are meant to illustrate the invention, and not to limitit in any way. One of skill in the art will appreciate that the stepstaken in each example may in some circumstances be performed in adifferent order to achieve the same result. One of skill in the art willalso understand that a different set of steps may also produce the sameresulting glycan. Further, the preferred remodeled glycan is by no meansspecific to the expression system that the peptide is expressed in. Theremodeled glycans are only illustrative and one of skill in the art willknow how to take the principles from these examples and apply them topeptides produced in different expression systems to generate glycansnot specifically described herein.

C. Glycoconjugation, in General

The invention provides methods of preparing a conjugate of aglycosylated or an unglycosylated peptide. The conjugates of theinvention are formed between peptides and diverse species such aswater-soluble polymers, therapeutic moieties, diagnostic moieties,targeting moieties and the like. Also provided are conjugates thatinclude two or more peptides linked together through a linker arm, i.e.,multifunctional conjugates. The multi-functional conjugates of theinvention can include two or more copies of the same peptide or acollection of diverse peptides with different structures, and/orproperties.

The conjugates of the invention are formed by the enzymatic attachmentof a modified sugar to the glycosylated or unglycosylated peptide. Themodified sugar, when interposed between the peptide and the modifyinggroup on the sugar becomes what is referred to herein as “an intactglycosyl linking group.” Using the exquisite selectivity of enzymes,such as glycosyltransferases, the present method provides peptides thatbear a desired group at one or more specific locations. Thus, accordingto the present invention, a modified sugar is attached directly to aselected locus on the peptide chain or, alternatively, the modifiedsugar is appended onto a carbohydrate moiety of a peptide. Peptides inwhich modified sugars are linked to both a peptide carbohydrate anddirectly to an amino acid residue of the peptide backbone are alsowithin the scope of the present invention.

In contrast to known chemical and enzymatic peptide elaborationstrategies, the methods of the invention make it possible to assemblepeptides and glycopeptides that have a substantially homogeneousderivatization pattern; the enzymes used in the invention are generallyselective for a particular amino acid residue or combination of aminoacid residues of the peptide or particular glycan structure. The methodsare also practical for large-scale production of modified peptides andglycopeptides. Thus, the methods of the invention provide a practicalmeans for large-scale preparation of peptides having preselectedsubstantially uniform derivatization patterns. The methods areparticularly well suited for modification of therapeutic peptides,including but not limited to, peptides that are incompletelyglycosylated during production in cell culture cells (e.g., mammaliancells, insect cells, plant cells, fungal cells, yeast cells, orprokaryotic cells) or transgenic plants or animals.

The methods of the invention also provide conjugates of glycosylated andunglycosylated peptides with increased therapeutic half-life due to, forexample, reduced clearance rate, or reduced rate of uptake by the immuneor reticuloendothelial system (RES). Moreover, the methods of theinvention provide a means for masking antigenic determinants onpeptides, thus reducing or eliminating a host immune response againstthe peptide. Selective attachment of targeting agents can also be usedto target a peptide to a particular tissue or cell surface receptor thatis specific for the particular targeting agent. Moreover, there isprovided a class of peptides that are specifically modified with atherapeutic moiety.

1. The Conjugates

In a first aspect, the present invention provides a conjugate between apeptide and a selected moiety. The link between the peptide and theselected moiety includes an intact glycosyl linking group interposedbetween the peptide and the selected moiety. As discussed herein, theselected moiety is essentially any species that can be attached to asaccharide unit, resulting in a “modified sugar” that is recognized byan appropriate transferase enzyme, which appends the modified sugar ontothe peptide. The saccharide component of the modified sugar, wheninterposed between the peptide and a selected moiety, becomes an “intactglycosyl linking group.” The glycosyl linking group is formed from anymono- or oligo-saccharide that, after modification with a selectedmoiety, is a substrate for an appropriate transferase.

The conjugates of the invention will typically correspond to the generalstructure:

in which the symbols a, b, c, d and s represent a positive, non-zerointeger; and t is either 0 or a positive integer. The “agent” is atherapeutic agent, a bioactive agent, a detectable label, water-solublemoiety or the like. The “agent” can be a peptide, e.g., enzyme,antibody, antigen, etc. The linker can be any of a wide array of linkinggroups, infra. Alternatively, the linker may be a single bond or a “zeroorder linker.” The identity of the peptide is without limitation.Exemplary peptides are provided in FIG. 28.

In an exemplary embodiment, the selected moiety is a water-solublepolymer. The water-soluble polymer is covalently attached to the peptidevia an intact glycosyl linking group. The glycosyl linking group iscovalently attached to either an amino acid residue or a glycosylresidue of the peptide. Alternatively, the glycosyl linking group isattached to one or more glycosyl units of a glycopeptide. The inventionalso provides conjugates in which the glycosyl linking group is attachedto both an amino acid residue and a glycosyl residue.

In addition to providing conjugates that are formed through anenzymatically added intact glycosyl linking group, the present inventionprovides conjugates that are highly homogenous in their substitutionpatterns. Using the methods of the invention, it is possible to formpeptide conjugates in which essentially all of the modified sugarmoieties across a population of conjugates of the invention are attachedto multiple copies of a structurally identical amino acid or glycosylresidue. Thus, in a second aspect, the invention provides a peptideconjugate having a population of water-soluble polymer moieties, whichare covalently linked to the peptide through an intact glycosyl linkinggroup. In a preferred conjugate of the invention, essentially eachmember of the population is linked via the glycosyl linking group to aglycosyl residue of the peptide, and each glycosyl residue of thepeptide to which the glycosyl linking group is attached has the samestructure.

Also provided is a peptide conjugate having a population ofwater-soluble polymer moieties covalently linked thereto through anintact glycosyl linking group. In a preferred embodiment, essentiallyevery member of the population of water soluble polymer moieties islinked to an amino acid residue of the peptide via an intact glycosyllinking group, and each amino acid residue having an intact glycosyllinking group attached thereto has the same structure.

The present invention also provides conjugates analogous to thosedescribed above in which the peptide is conjugated to a therapeuticmoiety, diagnostic moiety, targeting moiety, toxin moiety or the likevia an intact glycosyl linking group. Each of the above-recited moietiescan be a small molecule, natural polymer (e.g., peptide) or syntheticpolymer.

In an exemplary embodiment, interleukin-2 (IL-2) is conjugated totransferrin via a bifunctional linker that includes an intact glycosyllinking group at each terminus of the PEG moiety (Scheme 1). Forexample, one terminus of the PEG linker is functionalized with an intactsialic acid linker that is attached to transferrin and the other isfunctionalized with an intact GalNAc linker that is attached to IL-2.

In another exemplary embodiment, EPO is conjugated to transferrin. Inanother exemplary embodiment, EPO is conjugated to glial derivedneurotropic growth factor (GDNF). In these embodiments, each conjugationis accomplished via a bifunctional linker that includes an intactglycosyl linking group at each terminus of the PEG moiety, asaforementioned. Transferrin transfers the protein across the blood brainbarrier.

As set forth in the Figures appended hereto, the conjugates of theinvention can include intact glycosyl linking groups that are mono- ormulti-valent (e.g., antennary structures), see, FIGS. 14-22. Theconjugates of the invention also include glycosyl linking groups thatare O-linked glycans originating from serine or threonine (FIG. 11).Thus, conjugates of the invention include both species in which aselected moiety is attached to a peptide via a monovalent glycosyllinking group. Also included within the invention are conjugates inwhich more than one selected moiety is attached to a peptide via amultivalent linking group. One or more proteins can be conjugatedtogether to take advantage of their biophysical and biologicalproperties.

In a still further embodiment, the invention provides conjugates thatlocalize selectively in a particular tissue due to the presence of atargeting agent as a component of the conjugate. In an exemplaryembodiment, the targeting agent is a protein. Exemplary proteins includetransferrin (brain, blood pool), human serum (HS)-glycoprotein (bone,brain, blood pool), antibodies (brain, tissue with antibody-specificantigen, blood pool), coagulation Factors V-XII (damaged tissue, clots,cancer, blood pool), serum proteins, e.g., α-acid glycoprotein, fetuin,α-fetal protein (brain, blood pool), β2-glycoprotein (liver,atherosclerosis plaques, brain, blood pool), G-CSF, GM-CSF, M-CSF, andEPO (immune stimulation, cancers, blood pool, red blood celloverproduction, neuroprotection), and albumin (increase in half-life).

In addition to the conjugates discussed above, the present inventionprovides methods for preparing these and other conjugates. Thus, in afurther aspect, the invention provides a method of forming a covalentconjugate between a selected moiety and a peptide. Additionally, theinvention provides methods for targeting conjugates of the invention toa particular tissue or region of the body.

In exemplary embodiments, the conjugate is formed between awater-soluble polymer, a therapeutic moiety, targeting moiety or abiomolecule, and a glycosylated or non-glycosylated peptide. Thepolymer, therapeutic moiety or biomolecule is conjugated to the peptidevia an intact glycosyl linking group, which is interposed between, andcovalently linked to both the peptide and the modifying group (e.g.,water-soluble polymer). The method includes contacting the peptide witha mixture containing a modified sugar and a glycosyltransferase forwhich the modified sugar is a substrate. The reaction is conducted underconditions sufficient to form a covalent bond between the modified sugarand the peptide. The sugar moiety of the modified sugar is preferablyselected from nucleotide sugars, activated sugars and sugars, which areneither nucleotides nor activated.

In one embodiment, the invention provides a method for linking two ormore peptides through a linking group. The linking group is of anyuseful structure and may be selected from straight-chain and branchedchain structures. Preferably, each terminus of the linker, which isattached to a peptide, includes a modified sugar (i.e., a nascent intactglycosyl linking group).

In an exemplary method of the invention, two peptides are linkedtogether via a linker moiety that includes a PEG linker. The constructconforms to the general structure set forth in the cartoon above. Asdescribed herein, the construct of the invention includes two intactglycosyl linking groups (i.e., s+t=1). The focus on a PEG linker thatincludes two glycosyl groups is for purposes of clarity and should notbe interpreted as limiting the identity of linker arms of use in thisembodiment of the invention.

Thus, a PEG moiety is functionalized at a first terminus with a firstglycosyl unit and at a second terminus with a second glycosyl unit. Thefirst and second glycosyl units are preferably substrates for differenttransferases, allowing orthogonal attachment of the first and secondpeptides to the first and second glycosyl units, respectively. Inpractice, the (glycosyl)¹-PEG-(glycosyl)² linker is contacted with thefirst peptide and a first transferase for which the first glycosyl unitis a substrate, thereby forming (peptide)¹-(glycosyl)¹-PEG-(glycosyl)².The first transferase and/or unreacted peptide is then optionallyremoved from the reaction mixture. The second peptide and a secondtransferase for which the second glycosyl unit is a substrate are addedto the (peptide)¹-(glycosyl)¹-PEG-(glycosyl)² conjugate, forming(peptide)¹-(glycosyl)¹-PEG-(glycosyl)²-(peptide)². Those of skill in theart will appreciate that the method outlined above is also applicable toforming conjugates between more than two peptides by, for example, theuse of a branched PEG, dendrimer, poly(amino acid), polysaccharide orthe like.

As noted previously, in an exemplary embodiment, interleukin-2 (IL-2) isconjugated to transferrin via a bifunctional linker that includes anintact glycosyl linking group at each terminus of the PEG moiety (Scheme1). The IL-2 conjugate has an in vivo half-life that is increased overthat of IL-2 alone by virtue of the greater molecular size of theconjugate. Moreover, the conjugation of IL-2 to transferrin serves toselectively target the conjugate to the brain. For example, one terminusof the PEG linker is functionalized with a CMP-sialic acid and the otheris functionalized with an UDP-GalNAc. The linker is combined with IL-2in the presence of a GalNAc transferase, resulting in the attachment ofthe GalNAc of the linker arm to a serine and/or threonine residue on theIL-2.

In another exemplary embodiment, transferrin is conjugated to a nucleicacid for use in gene therapy.

The processes described above can be carried through as many cycles asdesired, and is not limited to forming a conjugate between two peptideswith a single linker. Moreover, those of skill in the art willappreciate that the reactions functionalizing the intact glycosyllinking groups at the termini of the PEG (or other) linker with thepeptide can occur simultaneously in the same reaction vessel, or theycan be carried out in a step-wise fashion. When the reactions arecarried out in a step-wise manner, the conjugate produced at each stepis optionally purified from one or more reaction components (e.g.,enzymes, peptides).

A still further exemplary embodiment is set forth in Scheme 2. Scheme 2shows a method of preparing a conjugate that targets a selected protein,e.g., EPO, to bone and increases the circulatory half-life of theselected protein.

The use of reactive derivatives of PEG (or other linkers) to attach oneor more peptide moieties to the linker is within the scope of thepresent invention. The invention is not limited by the identity of thereactive PEG analogue. Many activated derivatives of poly(ethyleneglycol) are available commercially and in the literature. It is wellwithin the abilities of one of skill to choose, and synthesize ifnecessary, an appropriate activated PEG derivative with which to preparea substrate useful in the present invention. See, Abuchowski et al.Cancer Biochem. Biophys., 7: 175-186 (1984); Abuchowski et al., J. Biol.Chem., 252: 3582-3586 (1977); Jackson et al., Anal. Biochem., 165:114-127 (1987); Koide et al., Biochem Biophys. Res. Commun., 111:659-667 (1983)), tresylate (Nilsson et al., Methods Enzymol., 104: 56-69(1984); Delgado et al., Biotechnol. Appl. Biochem., 12: 119-128 (1990));N-hydroxysuccinimide derived active esters (Buckmann et al., Makromol.Chem., 182: 1379-1384 (1981); Joppich et al., Makromol. Chem., 180:1381-1384 (1979); Abuchowski et al., Cancer Biochem. Biophys., 7:175-186 (1984); Katreet al. Proc. Natl. Acad. Sci. U.S.A., 84: 1487-1491(1987); Kitamura et al., Cancer Res., 51: 4310-4315 (1991); Boccu etal., Z. Naturforsch., 38C: 94-99 (1983), carbonates (Zalipsky et al.,POLY(ETHYLENE GLYCOL) CHEMISTRY: BIOTECHNICAL AND BIOMEDICALAPPLICATIONS, Harris, Ed., Plenum Press, New York, 1992, pp. 347-370;Zalipsky et al., Biotechnol. Appl. Biochem., 15: 100-114 (1992);Veronese et al., Appl. Biochem. Biotech., 11: 141-152 (1985)),imidazolyl formates (Beauchamp et al., Anal. Biochem., 131: 25-33(1983); Berger et al., Blood, 71: 1641-1647 (1988)), 4-dithiopyridines(Woghiren et al., Bioconjugate Chem., 4: 314-318 (1993)), isocyanates(Byun et al., ASAIO Journal, M649-M-653 (1992)) and epoxides (U.S. Pat.No. 4,806,595, issued to Noishiki et al., (1989). Other linking groupsinclude the urethane linkage between amino groups and activated PEG.See, Veronese, et al., Appl. Biochem. Biotechnol., 11: 141-152 (1985).

In another exemplary embodiment in which a reactive PEG derivative isutilized, the invention provides a method for extending theblood-circulation half-life of a selected peptide, in essence targetingthe peptide to the blood pool, by conjugating the peptide to a syntheticor natural polymer of a size sufficient to retard the filtration of theprotein by the glomerulus (e.g., albumin). This embodiment of theinvention is illustrated in Scheme 3 in which erythropoietin (EPO) isconjugated to albumin via a PEG linker using a combination of chemicaland enzymatic modification.

Thus, as shown in Scheme 3, an amino acid residue of albumin is modifiedwith a reactive PEG derivative, such as X-PEG-(CMP-sialic acid), inwhich X is an activating group (e.g., active ester, isothiocyanate,etc). The PEG derivative and EPO are combined and contacted with atransferase for which CMP-sialic acid is a substrate. In a furtherillustrative embodiment, an E-amine of lysine is reacted with theN-hydroxysuccinimide ester of the PEG-linker to form the albuminconjugate. The CMP-sialic acid of the linker is enzymatically conjugatedto an appropriate residue on EPO, e.g., Gal, thereby forming theconjugate. Those of skill will appreciate that the above-describedmethod is not limited to the reaction partners set forth. Moreover, themethod can be practiced to form conjugates that include more than twoprotein moieties by, for example, utilizing a branched linker havingmore than two termini.

2. Modified Sugars

Modified glycosyl donor species (“modified sugars”) are preferablyselected from modified sugar nucleotides, activated modified sugars andmodified sugars that are simple saccharides that are neither nucleotidesnor activated. Any desired carbohydrate structure can be added to apeptide using the methods of the invention. Typically, the structurewill be a monosaccharide, but the present invention is not limited tothe use of modified monosaccharide sugars; oligosaccharides andpolysaccharides are useful as well.

The modifying group is attached to a sugar moiety by enzymatic means,chemical means or a combination thereof, thereby producing a modifiedsugar. The sugars are substituted at any position that allows for theattachment of the modifying moiety, yet which still allows the sugar tofunction as a substrate for the enzyme used to ligate the modified sugarto the peptide. In a preferred embodiment, when sialic acid is thesugar, the sialic acid is substituted with the modifying group at eitherthe 9-position on the pyruvyl side chain or at the 5-position on theamine moiety that is normally acetylated in sialic acid.

In certain embodiments of the present invention, a modified sugarnucleotide is utilized to add the modified sugar to the peptide.Exemplary sugar nucleotides that are used in the present invention intheir modified form include nucleotide mono-, di- or triphosphates oranalogs thereof. In a preferred embodiment, the modified sugarnucleotide is selected from a UDP-glycoside, CMP-glycoside, or aGDP-glycoside. Even more preferably, the modified sugar nucleotide isselected from an UDP-galactose, UDP-galactosamine, UDP-glucose,UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid, or CMP-NeuAc.N-acetylamine derivatives of the sugar nucleotides are also of use inthe method of the invention.

The invention also provides methods for synthesizing a modified peptideusing a modified sugar, e.g., modified-galactose, -facose, and -sialicacid. When a modified sialic acid is used, either a sialyltransferase ora trans-sialidase (for α2,3-linked sialic acid only) can be used inthese methods.

In other embodiments, the modified sugar is an activated sugar.Activated modified sugars, which are useful in the present invention aretypically glycosides which have been synthetically altered to include anactivated leaving group. As used herein, the term “activated leavinggroup” refers to those moieties, which are easily displaced inenzyme-regulated nucleophilic substitution reactions. Many activatedsugars are known in the art. See, for example, Vocadlo et al., InCARBOHYDRATE CHEMISTRY AND BIOLOGY, Vol. 2, Ernst et al. Ed., Wiley-VCHVerlag: Weinheim, Germany, 2000; Kodama et al., Tetrahedron Lett. 34:6419 (1993); Lougheed, et al., J. Biol. Chem. 274: 37717 (1999)).

Examples of activating groups (leaving groups) include fluoro, chloro,bromo, tosylate ester, mesylate ester, triflate ester and the like.Preferred activated leaving groups, for use in the present invention,are those that do not significantly sterically encumber the enzymatictransfer of the glycoside to the acceptor. Accordingly, preferredembodiments of activated glycoside derivatives include glycosylfluorides and glycosyl mesylates, with glycosyl fluorides beingparticularly preferred. Among the glycosyl fluorides, α-galactosylfluoride, α-mannosyl fluoride, α-glucosyl fluoride, α-fucosyl fluoride,α-xylosyl fluoride, α-sialyl fluoride, α-N-acetylglucosaminyl fluoride,α-N-acetylgalactosaminyl fluoride, β-galactosyl fluoride, β-mannosylfluoride, β-glucosyl fluoride, β-fucosyl fluoride, β-xylosyl fluoride,β-sialyl fluoride, β-N-acetylglucosaminyl fluoride andβ-N-acetylgalactosaminyl fluoride are most preferred.

By way of illustration, glycosyl fluorides can be prepared from the freesugar by first acetylating the sugar and then treating it withHF/pyridine. This generates the thermodynamically most stable anomer ofthe protected (acetylated) glycosyl fluoride (i.e., the α-glycosylfluoride). If the less stable anomer (i.e., the β-glycosyl fluoride) isdesired, it can be prepared by converting the peracetylated sugar withHBr/HOAc or with HCl to generate the anomeric bromide or chloride. Thisintermediate is reacted with a fluoride salt such as silver fluoride togenerate the glycosyl fluoride. Acetylated glycosyl fluorides may bedeprotected by reaction with mild (catalytic) base in methanol (e.g.NaOMe/MeOH). In addition, many glycosyl fluorides are commerciallyavailable.

Other activated glycosyl derivatives can be prepared using conventionalmethods known to those of skill in the art. For example, glycosylmesylates can be prepared by treatment of the fully benzylatedhemiacetal form of the sugar with mesyl chloride, followed by catalytichydrogenation to remove the benzyl groups.

In a further exemplary embodiment, the modified sugar is anoligosaccharide having an antennary structure. In a preferredembodiment, one or more of the termini of the antennae bear themodifying moiety. When more than one modifying moiety is attached to anoligosaccharide having an antennary structure, the oligosaccharide isuseful to “amplify” the modifying moiety; each oligosaccharide unitconjugated to the peptide attaches multiple copies of the modifyinggroup to the peptide. The general structure of a typical chelate of theinvention as set forth in the drawing above, encompasses multivalentspecies resulting from preparing a conjugate of the invention utilizingan antennary structure. Many antennary saccharide structures are knownin the art, and the present method can be practiced with them withoutlimitation.

Exemplary modifying groups are discussed below. The modifying groups canbe selected for one or more desirable property. Exemplary propertiesinclude, but are not limited to, enhanced pharmacokinetics, enhancedpharmacodynamics, improved biodistribution, providing a polyvalentspecies, improved water solubility, enhanced or diminishedlipophilicity, and tissue targeting.

D. Peptide Conjugates

a) Water-Soluble Polymers

The hydrophilicity of a selected peptide is enhanced by conjugation withpolar molecules such as amine-, ester-, hydroxyl- andpolyhydroxyl-containing molecules. Representative examples include, butare not limited to, polylysine, polyethyleneimine, poly(ethylene glycol)and poly(propyleneglycol). Preferred water-soluble polymers areessentially non-fluorescent, or emit such a minimal amount offluorescence that they are inappropriate for use as a fluorescent markerin an assay. Polymers that are not naturally occurring sugars may beused. In addition, the use of an otherwise naturally occurring sugarthat is modified by covalent attachment of another entity (e.g.,poly(ethylene glycol), poly(propylene glycol), poly(aspartate),biomolecule, therapeutic moiety, diagnostic moiety, etc.) is alsocontemplated. In another exemplary embodiment, a therapeutic sugarmoiety is conjugated to a linker arm and the sugar-linker arm issubsequently conjugated to a peptide via a method of the invention.

Methods and chemistry for activation of water-soluble polymers andsaccharides as well as methods for conjugating saccharides and polymersto various species are described in the literature. Commonly usedmethods for activation of polymers include activation of functionalgroups with cyanogen bromide, periodate, glutaraldehyde, biepoxides,epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides,trichlorotriazine, etc. (see, R. F. Taylor, (1991), PROTEINIMMOBILISATION, FUNDAMENTALS AND APPLICATIONS, Marcel Dekker, N.Y.; S.S. Wong, (1992), CHEMISTRY OF PROTEIN CONJUGATION AND CROSSLINKINNG, CRCPress,. Boca Raton; G. T. Hermanson et al., (1993), IMMOBILIZED AFFINITYLIGAND TECHNIQUES, Academic Press, N.Y.; Dunn, R. L., et al., Eds.POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series Vol.469, American Chemical Society, Washington, D.C. 1991).

Routes for preparing reactive PEG molecules and forming conjugates usingthe reactive molecules are known in the art. For example, U.S. Pat. No.5,672,662 discloses a water soluble and isolatable conjugate of anactive ester of a polymer acid selected from linear or branchedpoly(alkylene oxides), poly(oxyethylated polyols), poly(olefinicalcohols), and poly(acrylomorpholine), wherein the polymer has about 44or more recurring units.

U.S. Pat. No. 6,376,604 sets forth a method for preparing awater-soluble 1-benzotriazolylcarbonate ester of a water-soluble andnon-peptidic polymer by reacting a terminal hydroxyl of the polymer withdi(1-benzotriazoyl)carbonate in an organic solvent. The active ester isused to form conjugates with a biologically active agent such as aprotein or peptide.

WO 99/45964 describes a conjugate comprising a biologically active agentand an activated water soluble polymer comprising a polymer backbonehaving at least one terminus linked to the polymer backbone through astable linkage, wherein at least one terminus comprises a branchingmoiety having proximal reactive groups linked to the branching moiety,in which the biologically active agent is linked to at least one of theproximal reactive groups. Other branched poly(ethylene glycols) aredescribed in WO 96/21469, U.S. Pat. No. 5,932,462 describes a conjugateformed with a branched PEG molecule that includes a branched terminusthat includes reactive functional groups. The free reactive groups areavailable to react with a biologically active species, such as a proteinor peptide, forming conjugates between the poly(ethylene glycol) and thebiologically active species. U.S. Pat. No. 5,446,090 describes abifunctional PEG linker and its use in forming conjugates having apeptide at each of the PEG linker termini.

Conjugates that include degradable PEG linkages are described in WO99/34833; and WO 99/14259, as well as in U.S. Pat. No. 6,348,558. Suchdegradable linkages are applicable in the present invention.

Although both reactive PEG derivatives and conjugates formed using thederivatives are known in the art, until the present invention, it wasnot recognized that a conjugate could be formed between PEG (or otherpolymer) and another species, such as a peptide or glycopeptide, throughan intact glycosyl linking group.

Many water-soluble polymers are known to those of skill in the art andare useful in practicing the present invention. The term water-solublepolymer encompasses species such as saccharides (e.g., dextran, amylose,hyalouronic acid, poly(sialic acid), heparans, heparins, etc.); poly(amino acids), e.g., poly(glutamic acid); nucleic acids; syntheticpolymers (e.g., poly(acrylic acid), poly(ethers), e.g., poly(ethyleneglycol); peptides, proteins, and the like. The present invention may bepracticed with any water-soluble polymer with the sole limitation thatthe polymer must include a point at which the remainder of the conjugatecan be attached.

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and more WO 93/15189, and for conjugation between activatedpolymers and peptides, e.g. Coagulation Factor VIII (WO 94/15625),hemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No.4,412,989), ribonuclease and superoxide dismutase (Veronese at al., App.Biochem. Biotech. 11: 141-45 (1985)).

Preferred water-soluble polymers are those in which a substantialproportion of the polymer molecules in a sample of the polymer are ofapproximately the same molecular weight; such polymers are“homodisperse.”

The present invention is further illustrated by reference to apoly(ethylene glycol) conjugate. Several reviews and monographs on thefunctionalization and conjugation of PEG are available. See, forexample, Harris, Macronol. Chem. Phys. C25: 325-373 (1985); Scouten,Methods in Enzymology 135: 30-65 (1987); Wong et al., Enzyme Microb.Technol. 14: 866-874 (1992); Delgado et al., Critical Reviews inTherapeutic Drug Carrier Systems 9: 249-304 (1992); Zalipsky,Bioconjugate Chem. 6: 150-165 (1995); and Bhadra, et al., Pharmazie,57:5-29 (2002).

Poly(ethylene glycol) molecules suitable for use in the inventioninclude, but are not limited to, those described by the followingFormula 3:

-   R═H, alkyl, benzyl, aryl, acetal, OHC—, H₂N—CH₂CH₂—, HS—CH₂CH₂—,

-   X, Y, W, U (independently selected) ═O, S, NH, N—R′;-   R′, R″′ (independently selected)=alkyl, benzyl, aryl, alkyl aryl,    pyridyl, substituted aryl, arylalkyl, acylaryl;-   n=1 to 2000;-   m, q, p (independently selected)=0 to 20-   o=0 to 20;-   Z=HO, NH₂, halogen, S—R′″, activated esters,

-   -   -sugar-nucleotide, protein, imidazole, HOBT, tetrazole, halide;        and        V═HO, NH₂, halogen, S—R′″, activated esters, activated amides,        -sugar-nucleotide, protein.

In preferred embodiments, the poly(ethylene glycol) molecule is selectedfrom the following:

The poly(ethylene glycol) useful in forming the conjugate of theinvention is either linear or branched. Branched poly(ethylene glycol)molecules suitable for use in the invention include, but are not limitedto, those described by the following Formula:

-   R′, R″, R′″ (independently selected) ═H, alkyl, benzyl, aryl,    acetal, OHC—, H₂N—CH₂CH₂—, HS—CH²CH₂—, —(CH₂)_(q)CY-Z,    -sugar-nucleotide, protein, methyl, ethyl, heteroaryl, acylalkyl,    acylaryl, acylalkylaryl;-   X, Y, W, A, B (independently selected) ═O, S, NH, N—R′, (CH₂)₁;-   n, p (independently selected)=1 to 2000;-   m, q, o (independently selected)=0 to 20;-   Z=HO, NH₂, halogen, S—R′″, activated esters,

-   -   -sugar-nucleotide, protein;

-   V═HO, NH₂, halogen, S—R′″, activated esters, activated amides,    -   -sugar-nucleotide, protein.

The in vivo half-life, area under the curve, and/or residence time oftherapeutic peptides can also be enhanced with water-soluble polymerssuch as polyethylene glycol (PEG) and polypropylene glycol (PPG). Forexample, chemical modification of proteins with PEG (PEGylation)increases their molecular size and decreases their surface- andfunctional group-accessibility, each of which are dependent on the sizeof the PEG attached to the protein. This results in an improvement ofplasma half-lives and in proteolytic-stability, and a decrease inimmunogenicity and hepatic uptake (Chaffee et al. J. Clin. Invest. 89:1643-1651 (1992); Pyatak et al. Res. Commun. Chem. Pathol Pharmacol.29:113-127 (1980)). PEGylation of interleukin-2 has been reported toincrease its antitumor potency in vivo (Katre et al. Proc. Natl. Acad.Sci. USA. 84: 1487-1491 (1987)) and PEGylation of a F(ab′)2 derived fromthe monoclonal antibody A7 has improved its tumor localization (Kitamuraet al. Biochem. Biophys. Res. Commun. 28: 1387-1394 (1990)).

In one preferred embodiment, the in vivo half-life of a peptidederivatized with a water-soluble polymer by a method of the invention isincreased relevant to the in vivo half-life of the non-derivatizedpeptide. In another preferred embodiment, the area under the curve of apeptide derivatized with a water-soluble polymer using a method of theinvention is increased relevant to the area under the curve of thenon-derivatized peptide. In another preferred embodiment, the residencetime of a peptide derivatized with a water-soluble polymer using amethod of the invention is increased relevant to the residence time ofthe non-derivatized peptide. Techniques to determine the in vivohalf-life, the area under the curve and the residence time are wellknown in the art. Descriptions of such techniques can be found in J. G.Wagner, 1993, Pharmacokinetics for the Pharmaceutical Scientist,Technomic Publishing Company, Inc. Lancaster Pa.

The increase in peptide in vivo half-life is best expressed as a rangeof percent increase in this quantity. The lower end of the range ofpercent increase is about 40%, about 60%, about 80%, about 100%, about150% or about 200%. The upper end of the range is about 60%, about 80%,about 100%, about 150%, or more than about 250%.

In an exemplary embodiment, the present invention provides a PEGylatedfollicle stimulating hormone (Examples 23 and 24). In a furtherexemplary embodiment, the invention provides a PEGylated transferrin(Example 42).

Other exemplary water-soluble polymers of use in the invention include,but are not limited to linear or branched poly(alkylene oxides),poly(oxyethylated polyols), poly(olefinic alcohols), andpoly(acrylomorpholine), dextran, starch, poly(amino acids), etc.

b) Water-Insoluble Polymers

The conjugates of the invention may also include one or morewater-insoluble polymers. This embodiment of the invention isillustrated by the use of the conjugate as a vehicle with which todeliver a therapeutic peptide in a controlled manner. Polymeric drugdelivery systems are known in the art. See, for example, Dunn et al.,Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium SeriesVol. 469, American Chemical Society, Washington, D.C. 1991. Those ofskill in the art will appreciate that substantially any known drugdelivery system is applicable to the conjugates of the presentinvention.

Representative water-insoluble polymers include, but are not limited to,polyphosphazines, poly(vinyl alcohols), polyamides, polycarbonates,polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkyleneoxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes, poly(methyl methacrylate), poly(ethyl methacrylate),poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate),poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate)polyethylene, polypropylene, poly(ethylene glycol), poly(ethyleneoxide), poly (ethylene terephthalate), poly(vinyl acetate), polyvinylchloride, polystyrene, polyvinyl pyrrolidone, pluronics andpolyvinylphenol and copolymers thereof.

Synthetically modified natural polymers of use in conjugates of theinvention include, but are not limited to, alkyl celluloses,hydroxyalkyl celluloses, cellulose ethers, cellulose esters, andnitrocelluloses. Particularly preferred members of the broad classes ofsynthetically modified natural polymers include, but are not limited to,methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, celluloseacetate, cellulose propionate, cellulose acetate butyrate, celluloseacetate phthalate, carboxymethyl cellulose, cellulose triacetate,cellulose sulfate sodium salt, and polymers of acrylic and methacrylicesters and alginic acid.

These and the other polymers discussed herein can be readily obtainedfrom commercial sources such as Sigma Chemical Co. (St. Louis, Mo.),Polysciences (Warrenton, Pa.), Aldrich (Milwaukee, Wis.), Fluka(Ronkonkoma, N.Y.), and BioRad (Richmond, Calif.), or else synthesizedfrom monomers obtained from these suppliers using standard techniques.

Representative biodegradable polymers of use in the conjugates of theinvention include, but are not limited to, polylactides, polyglycolidesand copolymers thereof, poly(ethylene terephthalate), poly(butyricacid), poly(valeric acid), poly(lactide-co-caprolactone),poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, blends andcopolymers thereof. Of particular use are compositions that form gels,such as those including collagen, pluronics and the like.

The polymers of use in the invention include “hybrid” polymers thatinclude water-insoluble materials having within at least a portion oftheir structure, a bioresorbable molecule. An example of such a polymeris one that includes a water-insoluble copolymer, which has abioresorbable region, a hydrophilic region and a plurality ofcrosslinkable functional groups per polymer chain.

For purposes of the present invention, “water-insoluble materials”includes materials that are substantially insoluble in water orwater-containing environments. Thus, although certain regions orsegments of the copolymer may be hydrophilic or even water-soluble, thepolymer molecule, as a whole, does not to any substantial measuredissolve in water.

For purposes of the present invention, the term “bioresorbable molecule”includes a region that is capable of being metabolized or broken downand resorbed and/or eliminated through normal excretory routes by thebody. Such metabolites or break down products are preferablysubstantially non-toxic to the body.

The bioresorbable region may be either hydrophobic or hydrophilic, solong as the copolymer composition as a whole is not renderedwater-soluble. Thus, the bioresorbable region is selected based on thepreference that the polymer, as a whole, remains water-insoluble.Accordingly, the relative properties, i.e., the kinds of functionalgroups contained by, and the relative proportions of the bioresorbableregion, and the hydrophilic region are selected to ensure that usefulbioresorbable compositions remain water-insoluble.

Exemplary resorbable polymers include, for example, syntheticallyproduced resorbable block copolymers of poly(α-hydroxy-carboxylicacid)/poly(oxyalkylene, (see, Cohn et al., U.S. Pat. No. 4,826,945).These copolymers are not crosslinked and are water-soluble so that thebody can excrete the degraded block copolymer compositions. See, Youneset al., J. Biomed. Mater. Res. 21: 1301-1316 (1987); and Cohn et al., J.Biomed. Mater. Res. 22: 993-1009 (1988).

Presently preferred bioresorbable polymers include one or morecomponents selected from poly(esters), poly(hydroxy acids),poly(lactones), poly(amides), poly(ester-amides), poly (amino acids),poly(anhydrides), poly(orthoesters), poly(carbonates),poly(phosphazines), poly(phosphoesters), poly(thioesters),polysaccharides and mixtures thereof. More preferably still, thebiosresorbable polymer includes a poly(hydroxy) acid component. Of thepoly(hydroxy) acids, polylactic acid, polyglycolic acid, polycaproicacid, polybutyric acid, polyvaleric acid and copolymers and mixturesthereof are preferred.

In addition to forming fragments that are absorbed in vivo(“bioresorbed”), preferred polymeric coatings for use in the methods ofthe invention can also form an excretable and/or metabolizable fragment.

Higher order copolymers can also be used in the present invention. Forexample, Casey et al., U.S. Pat. No. 4,438,253, which issued on Mar. 20,1984, discloses tri-block copolymers produced from thetransesterification of poly(glycolic acid) and an hydroxyl-endedpoly(alkylene glycol). Such compositions are disclosed for use asresorbable monofilament sutures. The flexibility of such compositions iscontrolled by the incorporation of an aromatic orthocarbonate, such astetra-p-tolyl orthocarbonate into the copolymer structure.

Other coatings based on lactic and/or glycolic acids can also beutilized. For example, Spinu, U.S. Pat. No. 5,202,413, which issued onApr. 13, 1993, discloses biodegradable multi-block copolymers havingsequentially ordered blocks of polylactide and/or polyglycolide producedby ring-opening polymerization of lactide and/or glycolide onto eitheran oligomeric diol or a diamine residue followed by chain extension witha di-functional compound, such as, a diisocyanate, diacylchloride ordichlorosilane.

Bioresorbable regions of coatings useful in the present invention can bedesigned to be hydrolytically and/or enzymatically cleavable. Forpurposes of the present invention, “hydrolytically cleavable” refers tothe susceptibility of the copolymer, especially the bioresorbableregion, to hydrolysis in water or a water-containing environment.Similarly, “enzymatically cleavable” as used herein refers to thesusceptibility of the copolymer, especially the bioresorbable region, tocleavage by endogenous or exogenous enzymes.

When placed within the body, the hydrophilic region can be processedinto excretable and/or metabolizable fragments. Thus, the hydrophilicregion can include, for example, polyethers, polyalkylene oxides,polyols, poly(vinyl pyrrolidine), poly(vinyl alcohol), poly(alkyloxazolines), polysaccharides, carbohydrates, peptides, proteins andcopolymers and mixtures thereof. Furthermore, the hydrophilic region canalso be, for example, a poly(alkylene) oxide. Such poly(alkylene) oxidescan include, for example, poly(ethylene) oxide, poly(propylene) oxideand mixtures and copolymers thereof.

Polymers that are components of hydrogels are also useful in the presentinvention. Hydrogels are polymeric materials that are capable ofabsorbing relatively large quantities of water. Examples of hydrogelforming compounds include, but are not limited to, polyacrylic acids,sodium carboxymethylcellulose, polyvinyl alcohol, polyvinyl pyrrolidine,gelatin, carrageenan and other polysaccharides,hydroxyethylenemethacrylic acid (HEMA), as well as derivatives thereof,and the like. Hydrogels can be produced that are stable, biodegradableand bioresorbable. Moreover, hydrogel compositions can include subunitsthat exhibit one or more of these properties.

Bio-compatible hydrogel compositions whose integrity can be controlledthrough crosslinking are known and are presently preferred for use inthe methods of the invention. For example, Hubbell et al., U.S. Pat. No.5,410,016, which issued on Apr. 25, 1995 and U.S. Pat. No. 5,529,914,which issued on June 25, 1996, disclose water-soluble systems, which arecrosslinked block copolymers having a water-soluble central blocksegment sandwiched between two hydrolytically labile extensions. Suchcopolymers are further end-capped with photopolymerizable acrylatefunctionalities. When crosslinked, these systems become hydrogels. Thewater soluble central block of such copolymers can include poly(ethyleneglycol); whereas, the hydrolytically labile extensions can be apoly(α-hydroxy acid), such as polyglycolic acid or polylactic acid. See,Sawhney et al., Macromolecules 26: 581-587 (1993).

In another preferred embodiment, the gel is a thermoreversible gel.Thermoreversible gels including components, such as pluronics, collagen,gelatin, hyalouronic acid, polysaccharides, polyurethane hydrogel,polyurethane-urea hydrogel and combinations thereof are presentlypreferred.

In yet another exemplary embodiment, the conjugate of the inventionincludes a component of a liposome. Liposomes can be prepared accordingto methods known to those skilled in the art, for example, as describedin Eppstein et al., U.S. Pat. No. 4,522,811, which issued on Jun. 11,1985. For example, liposome formulations may be prepared by dissolvingappropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine,stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, andcholesterol) in an inorganic solvent that is then evaporated, leavingbehind a thin film of dried lipid on the surface of the container. Anaqueous solution of the active compound or its pharmaceuticallyacceptable salt is then introduced into the container. The container isthen swirled by hand to free lipid material from the sides of thecontainer and to disperse lipid aggregates, thereby forming theliposomal suspension.

The above-recited microparticles and methods of preparing themicroparticles are offered by way of example and they are not intendedto define the scope of microparticles of use in the present invention.It will be apparent to those of skill in the art that an array ofmicroparticles, fabricated by different methods, are of use in thepresent invention.

c) Biomolecules

In another preferred embodiment, the modified sugar bears a biomolecule.In still further preferred embodiments, the biomolecule is a functionalprotein, enzyme, antigen, antibody, peptide, nucleic acid (e.g., singlenucleotides or nucleosides, oligonucleotides, polynucleotides andsingle- and higher-stranded nucleic acids), lectin, receptor or acombination thereof.

Some preferred biomolecules are essentially non-fluorescent, or emitsuch a minimal amount of fluorescence that they are inappropriate foruse as a fluorescent marker in an assay. Other biomolecules may befluorescent. The use of an otherwise naturally occurring sugar that ismodified by covalent attachment of another entity (e.g., PEG,biomolecule, therapeutic moiety, diagnostic moiety, etc.) isappropriate. In an exemplary embodiment, a sugar moiety, which is abiomolecule, is conjugated to a linker arm and the sugar-linker armcassette is subsequently conjugated to a peptide via a method of theinvention.

Biomolecules useful in practicing the present invention can be derivedfrom any source. The biomolecules can be isolated from natural sourcesor they can be produced by synthetic methods. Peptides can be naturalpeptides or mutated peptides. Mutations can be effected by chemicalmutagenesis, site-directed mutagenesis or other means of inducingmutations known to those of skill in the art. Peptides useful inpracticing the instant invention include, for example, enzymes,antigens, antibodies and receptors. Antibodies can be either polyclonalor monoclonal; either intact or fragments. The peptides are optionallythe products of a program of directed evolution.

Both naturally derived and synthetic peptides and nucleic acids are ofuse in conjunction with the present invention; these molecules can beattached to a sugar residue component or a crosslinking agent by anyavailable reactive group. For example, peptides can be attached througha reactive amine, carboxyl, sulfhydryl, or hydroxyl group. The reactivegroup can reside at a peptide terminus or at a site internal to thepeptide chain. Nucleic acids can be attached through a reactive group ona base (e.g., exocyclic amine) or an available hydroxyl group on a sugarmoiety (e.g., 3′- or 5′-hydroxyl). The peptide and nucleic acid chainscan be further derivatized at one or more sites to allow for theattachment of appropriate reactive groups onto the chain. See, Chriseyet al. Nucleic Acids Res. 24: 3031-3039 (1996).

In a further preferred embodiment, the biomolecule is selected to directthe peptide modified by the methods of the invention to a specifictissue, thereby enhancing the delivery of the peptide to that tissuerelative to the amount of underivatized peptide that is delivered to thetissue. In a still further preferred embodiment, the amount ofderivatized peptide delivered to a specific tissue within a selectedtime period is enhanced by derivatization by at least about 20%, morepreferably, at least about 40%, and more preferably still, at leastabout 100%. Presently, preferred biomolecules for targeting applicationsinclude antibodies, hormones and ligands for cell-surface receptors.Exemplary targeting biomolecules include, but are not limited to, anantibody specific for the transferrin receptor for delivery of themolecule to the brain (Penichet et al., 1999, J. Immunol. 163:4421-4426;Pardridge, 2002, Adv. Exp. Med. Biol. 513:397-430), a peptide thatrecognizes the vasculature of the prostate (Arap et al., 2002, PNAS99:1527-1531), and an antibody specific for lung caveolae (McIntosh etal., 2002, PNAS 99:1996-2001).

In a presently preferred embodiment, the modifying group is a protein.In an exemplary embodiment, the protein is an interferon. Theinterferons are antiviral glycoproteins that, in humans, are secreted byhuman primary fibroblasts after induction with virus or double-strandedRNA. Interferons are of interest as therapeutics, e.g., antivirals andtreatment of multiple sclerosis. For references discussing interferon-β,see, e.g., Yu, et al., J. Neuroimmunol., 64(1):91-100 (1996); Schmidt,J., J. Neurosci. Res., 65(1):59-67 (2001); Wender, et al., FoliaNeuropathol., 39(2):91-93 (2001); Martin, et al, Springer Semin.Immunopathol., 18(1): 1-24 (1996); Takane, et al., J. Pharmacol. Exp.Ther., 294(2):746-752 (2000); Sburlati, et al., Biotechnol. Prog.,14:189-192 (1998); Dodd, et al., Biochimica et Biophysica Acta,787:183-187 (1984); Edelbaum, et al., J. Interferon Res., 12:449-453(1992); Conradt, et al., J. Biol. Chem., 262(30):14600-14605 (1987);Civas, et al., Eur. J. Biochem., 173:311-316 (1988); Demolder, et al, J.Biotechnol., 32:179-189 (1994); Sedmak, et al., J. Interferon Res.,9(Suppl 1):S61-S65 (1989); Kagawa, et al., J. Biol. Chem.,263(33):17508-17515 (1988); Hershenson, et al., U.S. Pat. No. 4,894,330;Jayaram, et al., J. Interferon Res., 3(2):177-180 (1983); Menge, et al.,Develop. Biol. Standard., 66:391-401 (1987); Vonk, et al, J. InterferonRes., 3(2): 169-175 (1983); and Adolf, et al., J. Interferon Res.,10:255-267 (1990). For references relevant to interferon-α, see, Asano,et al., Eur. J. Cancer, 27(Suppl 4):S21-S25 (1991); Nagy, et al.,Anticancer Research, 8(3):467-470 (1988); Dron, et al., J. Biol. Regul.Homeost. Agents, 3(1):13-19 (1989); Habib, et al., Am. Surg.,67(3):257-260 (3/2001); and Sugyiama, et al., Eur. J. Biochem.,217:921-927 (1993).

In an exemplary interferon conjugate, interferon β is conjugated to asecond peptide via a linker arm. The linker arm includes an intactglycosyl linking group through which it is attached to the secondpeptide via a method of the invention. The linker arm also optionallyincludes a second intact glycosyl linking group, through which it isattached to the interferon.

In another exemplary embodiment, the invention provides a conjugate offollicle stimulating hormone (FSH). FSH is a glycoprotein hormone. See,for example, Saneyoshi, et al., Biol. Reprod., 65:1686-1690 (2001);Hakola, et al., J. Endocrinol., 158:441-448 (1998); Stanton, et al.,Mol. Cell. Endocrinol., 125:133-141 (1996); Walton, et al, J. Clin.Endocrinol. Metab., 86(8):3675-3685 (08/2001); Ulloa-Aguirre, et al,Endocrine, 11(3):205-215 (12/1999); Castro-Fernández, et al. I, J. Clin.Endocrinol. Matab., 85(12):4603-4610 (2000); Prevost, Rebecca R.,Pharmacotherapy, 18(5):1001-1010 (1998); Linskens, et al., The FASEBJournal, 13:639-645 (04/1999); Butnev, et al., Biol. Reprod., 58:458-469(1998); Muyan, et al., Mol. Endo., 12(5):766-772 (1998); Min, et al.,Endo. J., 43(5):585-593 (1996); Boime, et al., Recent Progress inHormone Research, 34:271-289 (1999); and Rafferty, et al., J. Endo.,145:527-533 (1995). The FSH conjugate can be formed in a manner similarto that described for interferon.

In yet another exemplary embodiment, the conjugate includeserythropoietin (EPO). EPO is known to mediate response to hypoxia and tostimulate the production of red blood cells. For pertinent references,see, Cerami, et al., Seminars in Oncology, 28(2)(Suppl 8):66-70(04/2001). An exemplary EPO conjugate is formed analogously to theconjugate of interferon.

In a further exemplary embodiment, the invention provides a conjugate ofhuman granulocyte colony stimulating factor (G-CSF). G-CSF is aglycoprotein that stimulates proliferation, differentiation andactivation of neutropoietic progenitor cells into functionally matureneutrophils. Injected G-CSF is known to be rapidly cleared from thebody. See, for example, Nohynek, et al., Cancer Chemother. Pharmacol.,39:259-266 (1997); Lord, et al., Clinical Cancer Research,7(7):2085-2090 (07/2001); Rotondaro, et al., Molecular Biotechnology,11(2): 117-128 (1999); and Bonig, et al., Bone Marrow Transplantation,28:259-264 (2001). An exemplary conjugate of G-CSF is prepared asdiscussed above for the conjugate of the interferons. One of skill inthe art will appreciate that many other proteins may be conjugated tointerferon using the methods and compositions of the invention,including but not limited to, the peptides listed in Tables 7 and 8(presented elsewhere herein) and FIG. 28, and in FIGS. 29-57, whereindividual modification schemes are presented.

In still a further exemplary embodiment, there is provided a conjugatewith biotin. Thus, for example, a selectively biotinylated peptide iselaborated by the attachment of an avidin or streptavidin moiety bearingone or more modifying groups.

In a further preferred embodiment, the biomolecule is selected to directthe peptide modified by the methods of the invention to a specificintracellular compartment, thereby enhancing the delivery of the peptideto that intracellular compartment relative to the amount ofunderivatized peptide that is delivered to the tissue. In a stillfurther preferred embodiment, the amount of derivatized peptidedelivered to a specific intracellular compartment within a selected timeperiod is enhanced by derivatization by at least about 20%, morepreferably, at least about 40%, and more preferably still, at leastabout 100%. In another particularly preferred embodiment, thebiomolecule is linked to the peptide by a cleavable linker that canhydrolyze once internalized. Presently, preferred biomolecules forintracellular targeting applications include transferrin,lactotransferrin (lactoferrin), melanotransferrin (p97), ceruloplasmin,and divalent cation transporter, as well as antibodies directed againstspecific vascular targets. Contemplated linkages include, but are notlimited to, protein-sugar-linker-sugar-protein,protein-sugar-linker-protein and multivalent forms thereof, andprotein-sugar-linker-drug where the drug includes small molecules,peptides, lipids, among others.

Site-specific and target-oriented delivery of therapeutic agents isdesirable for the purpose of treating a wide variety of human diseases,such as different types of malignancies and certain neurologicaldisorders. Such procedures are accompanied by fewer side effects and ahigher efficiacy of drug. Various principles have been relied on indesigning these delivery systems. For a review, see Garnett, AdvancedDrug Delivery Reviews 53:171-216 (2001).

One important consideration in designing a drug delivery system totarget tissues specifically. The discovery of tumor surface antigens hasmade it possible to develop therapeutic approaches where tumor cellsdisplaying definable surface antigens are specifically targeted andkilled. There are three main classes of therapeutic monoclonalantibodies (antibody) that have demonstrated effectiveness in humanclinical trials in treating malignancies: (1) unconjugated MAb, whicheither directly induces growth inhibition and/or apoptosis, orindirectly activates host defense mechanisms to mediate antitumorcytotoxicity; (2) drug-conjugated MAb, which preferentially delivers apotent cytotoxic toxin to the tumor cells and therefore minimizes thesystemic cytotoxicity commonly associated with conventionalchemotherapy; and (3) radioisotope-conjugated MAb, which delivers asterilizing dose of radiation to the tumor. See review by Reff et al.,Cancer Control 9:152-166 (2002).

In order to arm MAbs with the power to kill malignant cells, the MAbscan be connected to a toxin, which may be obtained from a plant,bacterial, or fungal source, to form chimeric proteins calledimmunotoxins. Frequently used plant toxins are divided into two classes:(1) holotoxins (or class II ribosome inactivating proteins), such asricin, abrin, mistletoe lectin, and modeccin, and (2) hemitoxins (classI ribosome inactivating proteins), such as pokeweed antiviral protein(PAP), saporin, Bryodin 1, bouganin, and gelonin. Commonly usedbacterial toxins include diphtheria toxin (DT) and Pseudomonas exotoxin(PE). Kreitman, Current Pharmaceutical Biotechnolog 2:313-325 (2001).Other toxins contemplated for use with the present invention include,but are not limited to, those in Table 2.

TABLE 2 Toxins. Chemical Structure Toxin Name/ Source/ CAS RN/Indication/ Activity (IC50 nM); Alternate ID Analogs Toxicity MechanismTumor Type

SW-163E/ 260794-24-9; Cancer and not reported 0.3 P388 Streptomyces spSNA 260794-25-0/ Antibacterial/ 0.2 A2780 15896/ SW-163C; low toxicity(mice ip) 0.4 KB SW-163E SW-163A; 1.6 colon SW-163B 1.3 HL-60

Thiocoraline/ 173046-02-1 Breast Cancer; DNA lung, colon, CNSMicromonospora marina Melanoma; Non-small Polymerase melanoma(actinomycete) lung cancer/ alpha not reported inhibitor (blocks cellprogression from G1 to

Trunkamide A¹/ 181758-83-8 Cancer/ not reported cell culture (IC50 inLissoclinum sp (aascidian) not reported micrograms/mL); 0.5 P388; 0.5A549; 0.5 HT-29; 1.0 MEL-28

Palauamine²/ 148717-58-2 Lung cancer/ not reported cell culture (IC50 inStylotella agminata LD50 (i.p. in mice) is 13 micrograms/mL); (sponge)mg/Kg 0.1 P388 0.2 A549 (lung) 2 HT-29 (colon) 10 KB

Halichondrin B/ 103614-76-2/ cancer/ antitubulin; NCI tumor panel;Halichondria Okadal, isohomohalic myelotoxicity dose cell cycle GI(50)from 50 nM to Axinell Carteri and hondrin B limiting (dogs, rats)inhibitor 0.1 nM; Phankell carteri (inhibits LC50's from 40 μM to(sponges)/ GTP binding 0.1 nM (many 0.1 to 25 NSC-609385 to tubulin) nM)

Isohomo-halichondrin B/ 157078-48-3/ melanoma, lung, CNS, antitubulin;IC50's in 0.1 nM range Halichondria Okadai, halichondrin colon, ovary/cell cycle (NCI tumor panel) Axinell Carteri and B not reportedinhibitor Phankell carteri (inhibits (sponges)/ GTP binding NSC-650467to tubulin)

Halichondrin B analogs/ 253128-15-3/ solid tumors/ tubulin cell culture(not semi-synthetic starting ER-076349; not reported binding reported);from Halichondria ER-086526; agent; animal models active Okadal, AxinellCarteri B-1793; disruption of (tumor regression and Phankell carteriE-7389 mitotic observed) in lymphoma, (sponges)/ spindles colon(multi-drug ER-076349; ER-086526; resistant). B-1793; E-7389

NK-130119/ 132707-68-7 antifungal and not reported 25 ng/mL colonStreptornyces anticancer/ 8.5 ng/mL lung bottropensis/ not reportedNK-130119

Tetrocarcin A/ 73666-84-9/ cancer/ inhibits the not reported notreported/ analogs are not reported anti- KF-67544 reported apoptoticfunctino of Bcl2

Gilvusniycin/ 195052-09-6 cancer/ not reported IC50's in ng/mL:Streptomyces QM16 not reported 0.08 P388 0.86 K562 (CML) 0.72 A431 (EC)0.75 MKN28 (GI); (for all < 1 nM)

IB-96212/ 220858-11-7/ Cancer and not reported IC50's in ng/mL: marineactinomycete/ IB-96212; Antibacterial/ 0.1 P388 IB-96212 1B-98214; notreported 1B-97227

BE-56384³/ 207570-04-5 cancer/ not reported IC50's in ng/mL:Streptoinyces Sp./ not reported 0.1 P388 BE-563 84 0.29 colon 26 34DLD-1 0.12 PC-13 0.12 MKM-45

Palmitoylrhizoxin/ 135819-69-1/ cancer/ tubulin not reportedsemi-synthetic; Rhizopus Analog of binds LDL; less binding chinensisrhizoxin cytotoxic than rhizoxin agent (cell cycle inhibitor)

Rhizoxin/ 95917-95-6; melanoma, lung, CNS, tubulin NCI tumor panel (NSCRhizopus chinensis/ 90996-54-6 colon, ovary, renal, binding 332598);WF-1360; NSC-332598; breast, head and neck/ agent (cell log G150's:FR-9002 16 Rapid Drug clearance; cycle 50 nM to 50 fM; High AUGcorrelates inhibitor) log LC50's: with high toxicity 50 μM to 0.5 nM(several cell lines at 50 fM).

Dolastatin-10/ 110417-88-4/ prostate, melanoma, tubulin NCI tumor panelDolabella auricularia (sea other leukemia/ binding (60 cell line; G150);hare)/ Dolistatins myelotoxicity (at greater (tubulin 25 nM to 1 pM(most < NSC-376 128 (ie. 15) and than 0.3 pM) aggregation) 1 nM) (threecell lines analogs μM)

soblidotin/ 149606-27-9/ cancer (pancreas, tubulin cell culture: colon,synthetic/ analogs esophageal colon, breast, binding melanoma, M5076TZT-1027; auristatin PE prepared lung, etc) / agent tumors, P388 with75- MTD was 1.8 mg/Kg 85% inhibition (dose (IV); toxicity not notreported) reported

Dolastatin-15/ not reported/ cancer/ Tubulin NCI tumor panel (60Dolabella auricularia (sea other not reported binding cell line; GI50);25 hare) Dolistatins (tubuline nM to 39 pM (most <1 (ie. 15) andaggregation) nM) (one cell line 2.5 analogs nM); most active in breast

Cemadotin⁴/ 1159776-69- melanoma/ tubulin NCI tumor panel (NCSSynthetic; Parent 9/ hypertension, myocardial binding D-669356); activein Dolastatin-15 was isolated many analogs ischemia and (tubulin breast,ovary, from Dolabella myelosuppression were aggregation) endometrial,sarcomas auricularia (sea hare)/ dose-limiting toxicities. and drugresistant cell LU-103793; NSCD- lines. Data not public. 669356

Epothilone A/ not reported/ cancer/ tubulin IC50's of; Synthetic orisolated from many analogs not reported binding 1.5 nM MCF-7 (breast)Sorangium cellulosuin (tubulin 27.1 nM MCF-7/ADR (myxococcales) strainpolymeriza- 2.1 nM KB-31 So ce90) tion) (melanoma) 3.2 nM HCT-116

Epothilone B/ 152044054-7/ Solid tumors (breast, tubulin IC50's of;Synthetic or isolated from many analogs ovarian, etc)/ binding 0.18 nMMCF-7 Sorangium cellulosuin well tolerated; t1/2 of (tubulin (breast)(myxococcales) strain So 2.5 hrs; partial polymeriza- 2.92 nM MCF-7/ADRce90)/ responses (phase I); tion) 0.19 nM KB-31 EPO-906 diarrhea majorside (melanoma) 0.42 nM HCT-116; effect. broad activity reported

Epothilone Analog/ not reported/ cancer/ tubulin IC50's of 0.30 toSynthetic or semi- hundreds of not reported binding 1.80 nM in varioussynthetic; Original lead, analogs (tubulin tumor cell lines; EpothiloneA, isolated polymeriza- active in drug resistant from Sorangium tion)cell lines cellulosun (myxococcales) strain So ce90)/ ZK-EPO

Epothilone D/ 189452-10-9/ Solid tumors (breast, tubulin NCI tumor panel(NSC- Epothilone D, isolated many analogs ovarian, etc)/ binding 703147;IC50); from Sorangium emesis and anemia; t1/2 (tubulin 0.19 nM KB-31cellulosum of 5-10 hrs. polymeriza- (melanoma) (myxococcales) strain Sotion) 0.42 nM HCT-116; ce90)/ broad activity reported KOS-862 StructureNot Identified Epothilone D analog⁵/ 189453-10-9/ Solid tumors; tubulinnot reported Synthetic or semi- hundreds of not reported bindingsynthetic; Original lead, analogs (tubulin Epothilone D, isolatedpolymeriza- from Sorangium tion) cellulosum (myxococcales) strain Soce90)/ KOS-166-24

Epothilone Analog/ not reported/ cancer; tubulin not reported Synthetic;Original lead, hundreds of not reported binding Epothilone A, isolatedanalogs (tubulin from Sorangium polymeriza- cellulosum tion)(myxococcales) strain So ce90)/ CGP-85715

Epothilone Analog/ 219989-84-1/ non-small cell Lung, tubulin NCI tumorPanel (NSC- Synthetic or semi- hundreds of breast, stomach tumor binding710428 & NSC- synthetic; Original lead, analogs (objective responses in(tubulin 710468); 8-32 nM Epothilone B, isolated breast ovarian andlung)/ polymerizati (NCI data not available) from Sorangium severtoxicity (fatigue, on) cellulosum anorexia, nauseas, (myxococcales)strain So vomiting, neuropathy ce90)/ myalgia) BMS-247550

Epothilone Analog/ not reported/ advanced cancers/ tubulin broadactivity with Synthetic or semi- hundreds of adverse events (diarrhea,binding IC50's of 0.7 to 10 nM synthetic; Original lead, analogs nausea,vomiting, (tubulin Epothilone B, isolated fatigue, neutropenia);polymeriza- from Sorangium t1/2 of 3.5 hrs; tion) cellulosum improvedwater (myxococcales) strain So solubility to BMS ce90)/ 247550.BMS-310705

Discodermolide/ 127943-53-7/ solid tumors/ tubulin Broad activity (A549-synthetic; orginally analogs less not reported; 100-fold stabilizingnsclung, prostate, P388, isolated from Discodermia potent increase inwater agent ovarian with IC50's dissoluta (deep water solubility overtaxol (similar to about 10 nM) including sponge); rare compound taxol)multi-drug resistant cell (7 mg per 0.5 Kg sponge/ lines; XAA-296

Chondramide D/ 172430-63-6 cancer/ tubulin 5 nM A-549 not reported notreported binding (epidermoid carcinoma) agent; actin 15 nM A-498(kidney) polymeriza- 14 nM A549 (lung) tion inhibitor 5 aM SK-OV-3(ovary) 3 nM U-937 (lymphoma)

Cryptophycin analogs 204990-60-3 solid tumors, colon tubulin broadactivity (lung, (including 52, 55 and and 186256- cancer/ polymeriza-breast, colon, leukemia) others)⁶/ 67-7/ Phase II studies halted tioninhibitor with IC50's of 2 to 40 Nostoc sp GSV 224 (blue- many potentbecause of severe pM; active against green algae) isolated analogstoxicity with one death multi-drug resistance Cryptophycin 1./ preparedat resulting from drug; cell lines (resistant to LY-355703; Ly-355702;Lilly MDR pump). NCI NSC-667642 tumor panel, G150's from 100 nM to 10pM; LC50's from 100 nM to 25 pM.

Cryptophycin 8/ 168482-36-8; solid tumors/ tubulin broad spectrumsemi-synthetic; starting 168482-40-4; not reported polymeriza-anticancer activity (cell material from Nostoc sp. 18665-94-1; tioninhibitor culture) including 124689-65-2; multi-drug resistant125546-14-7/ tumors cryptophycin 5, 15 and 35

Cryptophycin analogs⁷/ 219660-54-5/ solid tumors/ topoisomer- notreported synthetic; semi-synthetic, LY-404292 not reported aseinhibitors starting material from Nostoc sp./ LY-40429/

Arenastatin A analogs⁸/ not reported/ cancer/ inhibits 8.7 nM (5 pg/mL)KB Dysidea arenaria (marine analogs not reported tubulin(nasopharyngeal); NCI sponge)/ prepared polymeriza- tumor panel(GI50's); Cryptophycin B; NSC- tion 100 pM to3pM 670038

Phomopsin A/ not reported Liver cancer (not as tubulin potent anticancerDiaporte toxicus or potent in other cancers)/ binding activityespecially Phomopsin not reported agent against liver cancerleptostromifomils (fungi)

Curacin A and analogs/ 155233-30-0/ Cancer/ Tubulin broad activity(cancer Lyngbya majuscula (blue analogs have not reported binding celllines); 1-29 nM green cyanobacterium) been prepared agent

Hemiasterlins A & B not reported/ Cancer/ Antimitotic broad activity:and analogs^(9/) criamide A & not reported agent 0.3-3 nM MCF7Cymbastela sp. B; (tubulin (breast); geodiamiolid- binding 0.4 ng/mLP388 G agent)

Spongistatins (1-9)¹⁰/ 149715-96-8; cancer/ tubulin Most potentcompounds Spirastrell spinispirulifera 158734-18-0; not reported bindingever tested in NCI panel (sea sponge) 158681-42-6; agent cell line (meanG150's 158080-65-0; of 0.1 nM; 150642-07-2; Spongistatin-1 GI50's153698-80-7; of 0.025-0.035 nM with 153745-94-9; extremely potent150624-44-5; activity against a subset 158734-19-1/ of highly otherchemoresistant tumor spongistatins types

Maytansine/ 35846-53-8/ cancer/ tubulin Broad Activity in NCL Maytenussp./ other related severe toxicity binding tumor panel (NSC NSC-153858macrolides agent (causes 153858; NSC-153858); extensive NCI tumor panel,disassembly GI50's from 3 pM to of the 0.1 pM; LC50's from microtubule250 μM to 10 pM. Two and totally different experimen's prevents gavevery different tubulin potencies. spiralizaiton)

Maytansine-IgG(EGFR not reported/ breast, head and neck, EGFR notreported directed)-conjugate¹¹/ other related Squamous cell binding andsemi-synthetic; starting macrolides carcinoma/ tubulin material fromMaytenus not reported binding sp.

Maytansine-IgG(CD56 not reported/ Neuroendocrine, small- CD56antigen-specific antigen)-conjugate ¹², 3.5 other related cell lung,carcinoma/ binding and cytotoxicity (cell drug molecules per IgG/macrolides mild toxicity (fatigue, tubulin culture; epidermal,semi-synthetic; starting nausea, headaches and binding breast, renalovarian material from Maytenus mild peripheral colon) with IC50's ofsp./ neuropathy); no 10-40 pM; animal huN901-DM1 hematological toxicity;studies (miceSCLC MTD 60 mg/Kg, I.V., tumor-alone and in weekly for 4weeks; only combination with taxol stable disease reported or cisplatincompletely (humans) eliminated tumors).

Maytansine-IgG(CEA not reported/ non-small-cell lung, CEA bindingantigen-specific antigen)-conjugate¹³, 4 other related carcinomapancreas, and tubulin cytotoxicity (cell drug molecules per IgG/macrolides lung, colon/ binding culture; epidermal, semi-synthetic;starting mild toxicity (fatigue, breast, renal ovarian material fromMaytenus nausea, headaches and colon) with IC50's of sp./ mildperipheral 10-40 pM; animal C424-DM1 neuropathy); pancreatic studies(mice: lipase elevated; MTD 88 melanoma [COLO- mg/Kg, LV., every 21205]-alone and in days; only stable disease combination with taxolreported (humans); tl/2 or cisplatin completely was 44 hr. eliminatedtumors);

Geldanamycin/ 30562-34-6/ cancer/ binds Hsp 90 NCI tumor panel (cellStreptonzyces natural not reported chaperone culture); 5.3 to 100hygroscopicus var. derivatives and inhibi's nM; most active in Geldanus/function colon, lung and NSC-2 12518; Antibiotic leukemia. NCI tumor U29135; NSC-122750 panel, GI50's from 10 μM to 0.1 nM; LC50's from 100 μMto 100 nM. Two assays with very different potencies.

Geldanamycin Analog/ 745747-14-7/ solid tumors/ binds Hsp 90 cellculture (not semi-synthetic; / Kosan, NCI Dose limiting toxicitieschaperone reported); animal CP-127374; 17-AAG; and UK (anemia, anorexia,and inhibi's models active (tumor NSC-330507 looking for diarrhea,nausea and function regression observed) in analogs with vomiting); t1/2(i.v.) is breast, ovary, longer tl/2 about 90 mm; no melanoma, colon.and oral objective responses activity; measured at 88 mg/Kg analogs(i.v. daily for 5 days, include: NSC- every 21 days); 255110; 682300;683661; 683663.

Geldanamycin analog/ not reported/ solid tumors/ binds Hsp 90 notreported semi-synthetic; / analogs not reported chaperone CP-202567prepared and inhibits function

Geldanamycin 345232-44-2/ breast/ binds Hsp 90 cell culture (noconjugates/ analogs not reported chaperone reported); animalsemi-synthetic;/ prepared and inhibits models performed LY-294002-GM;P13K-1- function; GM binds and inhibi's PI-3 kinase Structure NotReported Geldanamycin Analog/ not reported/ breast, prostate/ binds Hsp90 not reported not reported/ analogs not reported chaperone CNF-101prepared and inhibits function Structure Not Reported Geldanamycin- notreported/ prostate/ binds Hsp 90 not reported; conjugate testosteroneconjugate/ analogs not reported chaperone has a 15-fold selectivesemi-synthetic/ prepared and inhibits cytotoxicity for GMT-1 functionand androgen positive testosterone prostate cells receptors where it isintemalized

Podophyllotoxin/ 518-28-5/ Verruca vulgaris, tubulin broad activity(cell Podophyllum sp. many analogs Condyloma/ inhibitor and culture)with IC50's in severe toxicity when topoisomer- μM range given i.v. ors.c. ase inhibitor

esperamicin-A1/ 99674-26-7 cancer/ DNA highly patent activity not known/not reported (suspected cleaving (cell culture); animal BBM-1G75A1; BMY-severe toxicity) agent models highly potent 28175; GGM-1675 with optimaldose of 0.16 micrograms/Kg

C-1027¹⁴/ 120177-69-7 cancer (examined DNA extremely potent (cellStreptomyces setonii C- hepatoma, breast, lung cleaving culture) IC50'sin pM 1027/ and leukemia/ agent and fM; conjugated to (2-1027 notreported antibodies the potency remains the same (ie. 5.5 to 42 pM);

Calieheamicin- 113440-58-7; AML DNA Kills CD33 + cells (HL- IgG(CD33antigen)- 220578-59-6/ mild toxicity cleaving 60, NOMO-1, andconjugate¹⁵/ several agent NKM-1) at 100 ng/mL; semi-synthetic: reportedin MDR cell lines are not Micromonospora patents effected by the drug.echinosporal gemtuzumab ozogamicin; mylotarg; WAY-CMA- 676; CMA-676;CDP- 771

Calicheamicin-IgG- 113440-58-7; cancer/ DNA TBD conjugates¹⁶/220578-59-6 not reported cleaving semi-synthetic: agent Microinonosporaechinospora

Calicheamicin- not reported cancer/ DNA all human cancer; data IgG(OBAIantigen) not reported cleaving not reported conjugate/ agentsemi-synthetic: Microinonospora echinospora/ OBA1-H8

Calicheamicin- not reported non-Hodgkin lymphoma, DNA all human cancer;data IgG(CD22 antigen) cancer/ cleaving not reported conjugate/ notreported agent semi-synthetic: Micromonospora echinospora/ CMC-544parially esterified polystyrene maleic acid copolymer (SMA) conjugatedto neocarzinostatin (NCS) Neocarzlnostatin¹⁷l 123760-07-6; liver cancerand brain DNA cell culture data not semi-synthetic; 9014-02-2 cancer/cleaving reported. Streptomyces not reported agent carconistaticus/Zinostatin stimalamer; YM-881; YM-16881 IgG (TES-23)-conjugated toneocarzinostatin Neocarzinostatin/ not reported solid tumors/ DNA cellculture data not not reported/ toxicity not reported; the cleavingreported. TES-23-NCS TES-23 antibody agent and (without anticancerinimunostim- agent) was as effective at ulator eliminating tumors as thedrug conjugated protein

Kedarcidin^(18/) 128512-40-3; cancer/ DNA cell culture (IC50's inStreptoalloteichus sp 128512-39-0/ not reported cleaving ng/mL), 0.4HCTL116; NOV strain L5856, ATCC chromophore agent 0.3 HCT116VP35; 53650/and protein 0.3 HCT116NM46; NSC-646276 conjugate 0.2 A2780; 1.3A2780/DDP. animal models in P388 and B-16 melanoma. NCI tumor panel,GI50's from 50 μM to 5 μM.

Eleutherobins/ 174545-76-7/ cancer/ tubulin similar potency to taxol;marine coral sarcodictyins not reported binding not effective against(marine coral) agent MDR cell lines

Bryostatin-1/ 83314-01-6 leukemia, melanoma, immnunostim- not reportedBugula neritina (marine lung, cancer/ ulant (TNF, bryosoan)/ myalgia;accumulated GMCSF, GMY-45618; NSC- toxicity; poor water etc); 339555solubility; dose limiting enhances cell toxicity kill by currentanticancer agents

FR-901228/ 128517-07-7 leukemia, T-cell histone In vitro cell lines (NCIChromobacterium lymphoma, cancer/ deacetylase tumor panel); violaceumnstrain 968/ toxic doses (LD50)6.4 inhiibitor IC50's of between 0.56NSC-63-176; FK-228 and 10 mg/Kg, ip and iv and 4.1 nM (breast,respectively; GI lung, gastric colon, toxicity, lymphoid leukemia)atrophy; dose limiting toxicity (human) 18 mg/Kg; t1/2 of 8 hrs (human)

Chlamydocin/ 53342-16-8 cancer/ histone not reported (cell not reportednot reported deacetylase culture); inhibitor inhibits histonedeacetylase at an IC50 of 1.3 nM

Phorboxazole A¹⁹/ 181377-57-1; leukemia, myeloma/ not reported NCI tumorpanel marine sponge 165689-31-6; not reported (induces (details notreported); 180911-824; apoptosis) IC50's of 1-10 nM. The 165883-76-1/inhibition values analogs (clonogenic growth of prepared human cancercells) at 10 nM ranged from 6.2 to > 99.9% against NALM-6 human B-lineage acute lymophoblastic leukemia cells, BT-20 breast cancer cellsand U373 glioblastoma cells, with the specified compound showinginhibition values in the range of 42.4 to > 99.9% against these celllines.; IC50's are nM for MDR cell lines.

Apicularen A/ 220757-06-2/ cancer/ not reported IC50's of 0.1 to 3Chondromyces robustus natural not reported ng/mL (KB-3-A, KB-derivatives Va, K562, HL60, U937, A498, A549, PV3 and SK-OV3)

Taxol/ 33069624/ cancer; breast, prostate, tubulin NCI tumor panel;Pacific yew and fungi/ many analogs ovary, colon, lung, head bindingG150's of 3 nM to 1 Paclitaxel; NSC-125973 & neck, etc./ agent μM;severe toxicity (grade III TGI 50 nM to 25 μM and IV)

Vitilevuamide/ 191681-63-7 cancer/ tubulin cell culture; IC50's ofDidemnum cuculliferum not reported binding 6-311 nM (panel of orPolysyncraton agent tumor cell lines lithostrotum HCT116 cells, A549cells, SK-MEL-5 cells A498 cells). The increase in lifespan (ILS) forCDF1 mice after ip injection of P388 tumor cells was in the range of −45to +70% over the dose range of 0.13 to 0.006 mg/kg.

Didemnin B/ 77327-05-0; non-Hodgkin's inhibits NCI 60-tumor panelTridideinnum solidum/ 77327-04-9; lymphoma, breast, protein (GI50's):100 nM to 50 NSC-2325319; IND 77327-06-1/ carcinoma, CNS, colon/synthesis via fM. 24505 other related Discontinued due to EF-1 Notpotent against natural cardiotoxicity; nausea, MDR cell lines. productsneuro-muscular toxicity and vomiting MTD 6.3 mg/Kg; toxicity preventedachieving a clinically signif. effect; rapidly cleared (t1/2 4.8 hrs

Leptomycin B/ 87081-35-4 NCI 60-tumor panel Streptomyces sp. strain(G150's): ATS 1287/ 8 μM to 1 μM; (L050): NSC-364372; elactocin 250 μMto 10 nM (several cell lines at 0.1 nM). Two testing results with verydifferent potencies.

Cryptopleurin/ NCI 60-tumor panel not known/ (G150's): 19 nM to 1NSC-19912 pM; (LC50): 40 μM to 10 nM (several cell lines at 1 pM).

Silicicolin/ 19186-35-7 NCI 60-tumor panel not known/ (GI50's): ~100 nMto 3 NSC-403148, nM; (LC50): 50 μM to deoxypodophyllotoxin, 10 nMdesoxypodophyllotoxin podophyllotoxin, deoxysilicicolin

Scillaren A/ 124-99-2 NCI 60-tumor panel not known/ (GI50's): 50 nM to0.1 NSC-7525; Gluco- nM; proscillaridin A; (LC50): 250 μM to 0.1Scillaren A nM

Cinerubin A-HCl/ not reported NCI 60-tumor panel not known/ (GI50's): 15nM to 10 NSC-243022; Cinerubin pM; (LC50): 100 μM A hydrochloride; to 6nM CL 86-F2 HCl; CL-86-F2-hydrochloride ¹WO-09739025; US-6025466²EP-00626383 30 November 1994 ³JP-10101676 ⁴W0-09705162; WO-09717364(dolastatin synthesis and analogs) ⁵Kosan licensed patent for Epothiloneanalogs from Sloan-Kettering; US 00185968 ⁶WO-09723211 ⁷WO-09723211⁸JP-08092232 ⁹W0-09633211 ¹⁰EP-00608111; EP-00632042; EP-00634414;WO-09748278 ¹¹EP-0042523S; JP-53124692 ¹²US 05416064; US-05208020;EP-00425235B ¹³EP-004252351 JP-53124692; US-06333410B1 ¹⁴JP-1104183¹⁵EP-00689845 ¹⁶EP 00689845 ¹⁷EP-00136791; EP-00087957 ¹⁸US 50001112; US5143906. ¹⁹WO-00136048

Conventional immunotoxins contain an MAb chemically conjugated to atoxin that is mutated or chemically modified to minimized binding tonormal cells. Examples include anti-B4-blocked ricin, targeting CD5; andRFB4-deglycosylated ricin A chain, targeting CD22. Recombinantimmunotoxins developed more recently are chimeric proteins consisting ofthe variable region of an antibody directed against a tumor antigenfused to a protein toxin using recombinant DNA technology. The toxin isalso frequently genetically modified to remove normal tissue bindingsites but retain its cytotoxicity. A large number of differentiationantigens, overexpressed receptors, or cancer-specific antigens have beenidentified as targets for immunotoxins, e.g., CD19, CD22, CD20, IL-2receptor (CD25), CD33, IL-4 receptor, EGF receptor and its mutants,ErB2, Lewis carbohydrate, mesothelin, transferrin receptor, GM-CSFreceptor, Ras, Bcr-Abl, and c-Kit, for the treatment of a variety ofmalignancies including hematopoietic cancers, glioma, and breast, colon,ovarian, bladder, and gastrointestinal cancers. See e.g., Brinkmann etal., Expert Opin. Biol. Ther. 1:693-702 (2001); Perentesis and Sievers,Hematology/Oncology Clinics of North America 15:677-701 (2001).

MAbs conjugated with radioisotope are used as another means of treatinghuman malignancies, particularly hematopoietic malignancies, with a highlevel of specificity and effectiveness. The most commonly used isotopesfor therapy are the high-energy-emitters, such as ¹³¹I and ⁹⁰Y.Recently, ²¹³Bi-labeled anti-CD33 humanized MAb has also been tested inphase I human clinical trials. Reff et al., supra.

A number of MAbs have been used for therapeutic purposes. For example,the use of rituximab (Rituxan™), a recombinant chimeric anti-CD20 MAb,for treating certain hematopoietic malignancies was approved by the FDAin 1997. Other MAbs that have since been approved for therapeutic usesin treating human cancers include: alemtuzumab (Campath-1H™), ahumanized rat antibody against CD52; and gemtuzumab ozogamicin(Mylotarg™), a calicheamicin-conjugated humanized mouse antCD33 MAb. TheFDA is also currently examining the safety and efficacy of several otherMAbs for the purpose of site-specific delivery of cytotoxic agents orradiation, e.g., radiolabeled Zevalin™ and Bexxar™, Reff et al., supra.

A second important consideration in designing a drug delivery system isthe accessibility of a target tissue to a therapeutic agent. This is anissue of particular concern in the case of treating a disease of thecentral nervous system (CNS), where the blood-brain barrier prevents thediffusion of macromolecules. Several approaches have been developed tobypass the blood-brain barrier for effective delivery of therapeuticagents to the CNS.

The understanding of iron transport mechanism from plasma to brainprovides a useful tool in bypassing the blood-brain barrier (BBB). Iron,transported in plasma by transferrin, is an essential component ofvirtually all types of cells. The brain needs iron for metabolicprocesses and receives iron through transferrin receptors located onbrain capillary endothelial cells via receptor-mediated transcytosis andendocytosis. Moos and Morgan, Cellular and Molecular Neurobiology20:77-95 (2000). Delivery systems based on transferrin-transferrinreceptor interaction have been established for the efficient delivery ofpeptides, proteins, and liposomes into the brain. For example, peptidescan be coupled with a Mab directed against the transferrin receptor toachieve greater uptake by the brain, Moos and Morgan, Supra. Similarly,when coupled with an MAb directed against the transferrin receptor, thetransportation of basic fibroblast growth factor (bFGF) across theblood-brain barrier is enhanced. Song et al., The Journal ofPharmacology and Experimental Therapeutics 301:605-610 (2002); Wu etal., Journal of Drug Targeting 10:239-245 (2002). In addition, aliposomal delivery system for effective transport of the chemotherapydrug, doxorubicin, into C6 glioma has been reported, where transferrinwas attached to the distal ends of liposomal PEG chains. Eavarone etal., J. Biomed. Mater. Res. 51:10-14 (2000). A number of US patents alsorelate to delivery methods bypassing the blood-brain barrier based ontransferrin-transferrin receptor interaction. See e.g., U.S. Pat. Nos.5,154,924; 5,182,107; 5,527,527; 5,833,988; 6,015,555.

There are other suitable conjugation partners for a pharmaceutical agentto bypass the blood-brain barrier. For example, U.S. Pat. Nos.5,672,683, 5,977,307 and WO 95/02421 relate to a method of delivering aneuropharmaceutical agent across the blood-brain barrier, where theagent is administered in the form of a fusion protein with a ligand thatis reactive with a brain capillary endothelial cell receptor; WO99/00150 describes a drug delivery system in which the transportation ofa drug across the blood-brain barrier is facilitated by conjugation withan MAb directed against human insulin receptor; WO 89/10134 describes achimeric peptide, which includes a peptide capable of crossing the bloodbrain barrier at a relatively high rate and a hydrophilic neuropeptideincapable of transcytosis, as a means of introducing hydrophilicneuropeptides into the brain; WO 01/60411 A1 provides a pharmaceuticalcomposition that can easily transport a pharmaceutically activeingredient into the brain. The active ingredient is bound to ahibernation-specific protein that is used as a conjugate, andadministered with a thyroid hormone or a substance promoting thyroidhormone production. In addition, an alternative route of drug deliveryfor bypassing the blood-brain barrier has been explored. For instance,intranasal delivery of therapeutic agents without the need forconjugation has been shown to be a promising alternative delivery method(Frey, 2002, Drug Delivery Technology, 2(5):46-49).

In addition to facilitating the transportation of drugs across theblood-brain barrier, transferrin-transferrin receptor interaction isalso useful for specific targeting of certain tumor cells, as many tumorcells overexpress transferrin receptor on their surface. This strategyhas been used for delivering bioactive macromolecules into K562 cellsvia a transferrin conjugate (Wellhoner et al., The Journal of BiologicalChemistry 266:4309-4314 (1991)), and for delivering insulin intoenterocyte-like Caco-2 cells via a transferrin conjugate (Shah and Shen,Journal of Pharmaceutical Sciences 85:1306-1311 (1996)).

Furthermore, as more becomes known about the functions of various irontransport proteins, such as lactotransferrin receptor,melanotransferrin, ceruloplasmin, and Divalent Cation Transporter andtheir expression pattern, some of the proteins involved in irontransport mechanism(e.g., melanotransferrin), or their fragments, havebeen found to be similarly effective in assisting therapeutic agentstransport across the blood-brain barrier or targeting specific tissues(WO 02/13843 A2, WO 02/13873 A2). For a review on the use of transferrinand related proteins involved in iron uptake as conjugates in drugdelivery, see Li and Qian, Medical Research Reviews 22:225-250 (2002).

The concept of tissue-specific delivery of therapeutic agents goesbeyond the interaction between transferrin and transferrin receptor ortheir related proteins. For example, a bone-specific delivery system hasbeen described in which proteins are conjugated with a bone-seekingaminobisphosphate for improved delivery of proteins to mineralizedtissue. Uludag and Yang, Bioteclnol. Prog. 18:604611 (2002). For areview on this topic, see Vyas et al., Critical Reviews in TherapeuticDrug Carrier System 18:1-76 (2001).

A variety of linkers may be used in the process of generatingbioconjugates for the purpose of specific delivery of therapeuticagents,. Suitable linkers include homo- and heterobifunctionalcross-linking reagents, which may be cleavable by, e.g., acid-catalyzeddissociation, or non-cleavable (see, e.g., Srinivasachar and Neville,Biochemnistry 28:2501-2509 (1989); Wellhoner et al., The Journal ofBiological Chemistry 266:4309-4314 (1991)). Interaction between manyknown binding partners, such as biotin and avidin/streptavidin, can alsobe used as a means to join a therapeutic agent and a conjugate partnerthat ensures the specific and effective delivery of the therapeuticagent. Using the methods of the invention, proteins may be used todeliver molecules to intracellular compartments as conjugates. Proteins,peptides, hormones, cytokines, small molecules or the like that bind tospecific cell surface receptors that are internalized after ligandbinding may be used for intracellular targeting of conjugatedtherapeutic compounds. Typically, the receptor-ligand complex isinternalized into intracellular vesicles that are delivered to specificcell compartments, including, but not limited to, the nucleus,mitochondria, golgi, ER, lysosome, and endosome, depending on theintracellular location targeted by the receptor. By conjugating thereceptor ligand with the desired molecule, the drug will be carried withthe receptor-ligand complex and be delivered to the intracellularcompartments normally targeted by the receptor. The drug can thereforebe delivered to a specific intracellular location in the cell where itis needed to treat a disease.

Many proteins may be used to target therapeutic agents to specifictissues and organs. Targeting proteins include, but are not limited to,growth factors (EPO, HGH, EGF, nerve growth factor, FGF, among others),cytokines (GM-CSF, G-CSF, the interferon family, interleukins, amongothers), hormones (FSH, LH, the steroid families, estrogen,corticosteroids, insulin, among others), serum proteins (albumin,lipoproteins, fetoprotein, human serum proteins, antibodies andfragments of antibodies, among others), and vitamins (folate, vitamin C,vitamin A, among others). Targeting agents are available that arespecific for receptors on most cells types.

Contemplated linkage configurations include, but are not limited to,protein-sugar-linker-sugar-protein and multivalent forms thereof,protein-sugar-linker-protein and multivalent forms thereof,protein-sugar-linker-therapeutic agent, where the therapeutic agentincludes, but are not limited to, small molecules, peptides and lipids.In some embodiments, a hydrolysable linker is used that can behydrolyzed once internalized. An acid labile linker can be used toadvantage where the protein conjugate is internalized into the endosomesor lysosomes which have an acidic pH. Once internalized into theendosome or lysosome, the linker is hydrolyzed and the therapeutic agentis released from the targeting agent.

In an exemplary embodiment, transferrin is conjugated via a linker to anenzyme or a nucleic acid vector that encoded the enzyme desired to betargeted to a cell that presents transferrin receptors in a patient. Thepatient could, for example, require enzyme replacement therapy for thatparticular enzyme. In particularly preferred embodiments, the enzyme isone that is lacking in a patient with a lysosomal storage disease (seeTable 5). Once in circulation, the transferrin-enzyme conjugate islinked to transferrin receptors and is internalized in early endosomes(Xing et al., 1998, Biochem. J. 336:667; Li et al., 2002, Trends inPharmcol. Sci. 23:206; Suhaila et al., 1998, J. Biol. Chem. 273:14355).Other contemplated targeting agents that are related to transferrininclude, but are not limited to, lactotransferrin (lactoferrin),melanotransferrin (p97), ceruloplasmin, and divalent cation transporter.

In another exemplary embodiment, transferrin-dystrophin conjugates wouldenter endosomes by the transferrin pathway. Once there, the dystrophinis released due to a hydrolysable linker which can then be taken to theintracellular compartment where it is required. This embodiment may beused to treat a patient with muscular dystrophy by supplementing agenetically defective dystrophin gene and/or protein with the functionaldystrophin peptide connected to the transferrin.

E. Therapeutic Moieties

In another preferred embodiment, the modified sugar includes atherapeutic moiety. Those of skill in the art will appreciate that thereis overlap between the category of therapeutic moieties andbiomolecules; many biomolecules have therapeutic properties orpotential.

The therapeutic moieties can be agents already accepted for clinical useor they can be drugs whose use is experimental, or whose activity ormechanism of action is under investigation. The therapeutic moieties canhave a proven action in a given disease state or can be onlyhypothesized to show desirable action in a given disease state. In apreferred embodiment, the therapeutic moieties are compounds, which arebeing screened for their ability to interact with a tissue of choice.Therapeutic moieties, which are useful in practicing the instantinvention include drugs from a broad range of drug classes having avariety of pharmacological activities. In some embodiments, it ispreferred to use therapeutic moieties that are not sugars. An exceptionto this preference is the use of a sugar that is modified by covalentattachment of another entity, such as a PEG, biomolecule, therapeuticmoiety, diagnostic moiety and the like. In an exemplary embodiment, anantisense nucleic acid moeity is conjugated to a linker arm which isattached to the targeting moiety. In another exemplary embodiment, atherapeutic sugar moiety is conjugated to a linker arm and thesugar-linker arm cassette is subsequently conjugated to a peptide via amethod of the invention.

Methods of conjugating therapeutic and diagnostic agents to variousother species are well known to those of skill in the art. See, forexample Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego,1996; and Dunn et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS,ACS Symposium Series Vol. 469, American Chemical Society, Washington,D.C. 1991.

In an exemplary embodiment, the therapeutic moiety is attached to themodified sugar via a linkage that is cleaved under selected conditions.Exemplary conditions include, but are not limited to, a selected pH(e.g., stomach, intestine, endocytotic vacuole), the presence of anactive enzyme (e.g., esterase, protease, reductase, oxidase), light,heat and the like. Many cleavable groups are known in the art. See, forexample, Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshiet al., J. Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J.Immunol., 124: 913-920 (1980); Bouizar et al., Eur. J. Biochem., 155:141-147 (1986); Park et al., J. Biol. Chem., 261: 205-210 (1986);Browning et al., J. Immunol., 143: 1859-1867 (1989).

Classes of useful therapeutic moieties include, for example,non-steroidal anti-inflammatory drugs (NSAIDS). The NSAIDS can, forexample, be selected from the following categories: (e.g., propionicacid derivatives, acetic acid derivatives, fenamic acid derivatives,biphenylcarboxylic acid derivatives and oxicams); steroidalanti-inflammatory drugs including hydrocortisone and the like;adjuvants; antihistaminic drugs (e.g., chlorpheniramine, triprolidine);antitussive drugs (e.g., dextromethorphan, codeine, caramiphen andcarbetapentane); antipruritic drugs (e.g., methdilazine andtrimeprazine); anticholinergic drugs (e.g., scopolamine, atropine,homatropine, levodopa); anti-emetic and antinauseant drugs (e.g.,cyclizine, meclizine, chlorpromazine, buclizine); anorexic drugs (e.g.,benzphetamine, phentermine, chlorphentermine, fenfluramine); centralstimulant drugs (e.g., amphetamine, methamphetamine, dextroamphetamineand methylphenidate); antiarrhythmic drugs (e.g., propanolol,procainamide, disopyiamide, quinidine, encainide); β-adrenergic blockerdrugs (e.g., metoprolol, acebutolol, betaxolol, labetalol and timolol);cardiotonic drugs (e.g., milrinone, arninone and dobutamine);antihypertensive drugs (e.g., enalapril, clonidine, hydralazine,minoxidil, guanadrel, guanethidine); diuretic drugs (e.g., amiloride andhydrochlorothiazide); vasodilator drugs (e.g., diltiazem, amiodarone,isoxsuprine, nylidrin, tolazoline and verapamil); vasoconstrictor drugs(e.g., dihydroergotamine, ergotamine and methylsergide); antiulcer drugs(e.g., ranitidine and cimetidine); anesthetic drugs (e.g., lidocaine,bupivacaine, chloroprocaine, dibucaine); antidepressant drugs (e.g.,imipramine, desipramine, arnitryptiline, nortryptiline); tranquilizerand sedative drugs (e.g., chlordiazepoxide, benacytyzine, benzquinamide,flurazepam, hydroxyzine, loxapine and promazine); antipsychotic drugs(e.g., chlorprothixene, fluphenazine, haloperidol, molindone,thioridazine and trifluoperazine); antimicrobial drugs (antibacterial,antifungal, antiprotozoal and antiviral drugs).

Classes of useful therapeutic moieties include adjuvants. The adjuvantscan, for example, be selected from keyhole lymphet hemocyaninconjugates, monophosphoryl lipid A, mycoplasma-derived lipopeptideMALP-2, cholera toxin B subunit, Escherichia coli heat-labile toxin,universal T helper epitope from tetanus toxoid, interleukin-12, CpGoligodeoxynucleotides, dimethyldioctadecylammonium bromide,cyclodextrin, squalene, aluminum salts, meningococcal outer membranevesicle (OMV), montanide ISA, TiterMax™ (available from Sigma, St. LouisMo.), nitrocellulose absorption, immune-stimulating complexes such asQuil A, Gerbu™ adjuvant (Gerbu Biotechnik, Kirchwald, Germany), threonylmuramyl dipeptide, thymosin alpha, bupivacaine, GM-CSF, IncompleteFreund's Adjuvant, MTP-PE/MF59 (Ciba/Geigy, Basel, Switzerland),polyphosphazene, saponin derived from the soapbark tree Quillajasaponaria, and Syntex adjuvant formulation (Biocine, Emeryville,Calif.), among others well known to those in the art.

Antimicrobial drugs which are preferred for incorporation into thepresent composition include, for example, pharmaceutically acceptablesalts of β-lactam drugs, quinolone drugs, ciprofloxacin, norfloxacin,tetracycline, erythromycin, amikacin, triclosan, doxycycline,capreomycin, chlorhexidine, chlortetracycline, oxytetracycline,clindamycin, ethambutol, hexamidine isothionate, metronidazole,pentamidine, gentamycin, kanamycin, lineomycin, methacycline,methenamine, minocycline, neomycin, netilmycin, paromomycin,streptomycin, tobramycin, miconazole and amantadine.

Other drug moieties of use in practicing the present invention includeantineoplastic drugs (e.g., antiandrogens (e.g., leuprolide orflutamide), cytocidal agents (e.g., adriamycin, doxorubicin, taxol,cyclophosphamide, busulfan, cisplatin, β-2-interferon) anti-estrogens(e.g., tamoxifen), antimetabolites (e.g., fluorouracil, methotrexate,mercaptopurine, thioguanine). Also included within this class areradioisotope-based agents for both diagnosis and therapy, and conjugatedtoxins, such as ricin, geldanamycin, mytansin, CC-1065, C-1027, theduocarmycins, calicheamycin and related structures and analoguesthereof, and the toxins listed in Table 2.

The therapeutic moiety can also be a hormone (e.g., medroxyprogesterone,estradiol, leuprolide, megestrol, octreotide or somatostatin); musclerelaxant drugs (e.g., cinnamedrine, cyclobenzaprine, flavoxate,orphenadrine, papaverine, mebeverine, idaverine, ritodrine,diphenoxylate, dantrolene and azumolen); antispasmodic drugs;bone-active drugs (e.g., diphosphonate and phosphonoalkylphosphinatedrug compounds); endocrine modulating drugs (e.g., contraceptives (e.g.,ethinodiol, ethinyl estradiol, norethindrone, mestranol, desogestrel,medroxyprogesterone), modulators of diabetes (e.g., glyburide orchlorpropamide), anabolics, such as testolactone or stanozolol,androgens (e.g., methyltestosterone, testosterone or fluoxymesterone),antidiuretics (e.g., desmopressin) and calcitonins).

Also of use in the present invention are estrogens (e.g.,diethylstilbesterol), glucocorticoids (e.g., triamcinolone,betamethasone, etc.) andprogesterones, such as norethindrone,ethynodiol, norethindrone, levonorgestrel; thyroid agents (e.g.,liothyronine or levothyroxine) or anti-thyroid agents (e.g.,methimazole); antihyperprolactinemic drugs (e.g., cabergoline); hormonesuppressors (e.g., danazol or goserelin), oxytocics (e.g.,methylergonovine or oxytocin) and prostaglandins, such as mioprostol,alprostadil or dinoprostone, can also be employed.

Other useful modifying groups include immunomodulating drugs (e.g.,antihistamines, mast cell stabilizers, such as lodoxamide and/orcromolyn, steroids (e.g., triamcinolone, beclomethazone, cortisone,dexamethasone, prednisolone, methylprednisolone, beclomethasone, orclobetasol), histamine H2 antagonists (e.g., famotidine, cimetidine,ranitidine), immunosuppressants (e.g., azathioprine, cyclosporin), etc.Groups with anti-inflammatory activity, such as sulindac, etodolac,ketoprofen and ketorolac, are also of use. Other drugs of use inconjunction with the present invention will be apparent to those ofskill in the art.

Classes of useful therapeutic moieties include, for example, antisensedrugs and also naked DNA. The antisense drugs can be selected from forexample Affinitak (ISIS, Carlsbad, Calif.) and Genasense™ (from Genta,Berkeley Heights, N.J.). Naked DNA can be delivered as a gene therapytherapeutic for example with the DNA encoding for example factors VIIIand IX for treatment of hemophilia disorders.

F. Preparation of Modified Sugars

Modified sugars useful in forming the conjugates of the invention arediscussed herein. The discussion focuses on preparing a sugar modifiedwith a water-soluble polymer for clarity of illustration. In particular,the discussion focuses on the preparation of modified sugars thatinclude a poly(ethylene glycol) moiety. Those of skill will appreciatethat the methods set forth herein are broadly applicable to thepreparation of modified sugars, therefore, the discussion should not beinterpreted as limiting the scope of the invention.

In general, the sugar moiety and the modifying group are linked togetherthrough the use of reactive groups, which are typically transformed bythe linking process into a new organic functional group or unreactivespecies. The sugar reactive functional group(s), is located at anyposition on the sugar moiety. Reactive groups and classes of reactionsuseful in practicing the present invention are generally those that arewell known in the art of bioconjugate chemistry. Currently favoredclasses of reactions available with reactive sugar moieties are those,which proceed under relatively mild conditions. These include, but arenot limited to nucleophilic substitutions (e.g., reactions of amines andalcohols with acyl halides, active esters), electrophilic substitutions(e.g., enamine reactions) and additions to carbon-carbon andcarbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alderaddition). These and other useful reactions are discussed in, forexample, Smith and March, ADVANCED ORGANIC CHEMISTRY, 5th Ed., JohnWiley & Sons, New York, 2001; Hermanson, BIOCONJUGATE TECHNIQUES,Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OFPROTEINS; Advances in Chernistry Series, Vol. 198, American ChemicalSociety, Washington, D.C., 1982.

Useful reactive functional groups pendent from a sugar nucleus ormodifing group include, but are not limited to:

(a) carboxyl groups and various derivatives thereof including, but notlimited to, N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters,acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl,alkenyl, alkynyl and aromatic esters;

(b) hydroxyl groups, which can be converted to, e.g., esters, ethers,aldehydes, etc.

(c) haloallyl groups, wherein the halide can be later displaced with anucleophilic group such as, for example, an amine, a carboxylate anion,thiol anion, carbanion, or an alkoxide ion, thereby resulting in thecovalent attachment of a new group at the functional group of thehalogen atom;

(d) dienophile groups, which are capable of participating in Diels-Alderreactions such as, for example, maleimido groups;

(e) aldehyde or ketone groups, such that subsequent derivatization ispossible via formation of carbonyl derivatives such as, for example,imines, hydrazones, semicarbazones or oximes, or via such mechanisms asGrignard addition or alkyllithium addition;

(f) sulfonyl halide groups for subsequent reaction with amines, forexample, to form sulfonamides;

(g) thiol groups, which can be, for example, converted to disulfides orreacted with alkyl and acyl halides;

(h) amine or sulfhydryl groups, which can be, for example, acylated,alkylated or oxidized;

(i) alkenes, which can undergo, for example, cycloadditions, acylation,Michael addition, etc; and

(j) epoxides, which can react with, for example, amines and hydroxylcompounds.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assemblethe reactive sugar nucleus or modifying group. Alternatively, a reactivefunctional group can be protected from participating in the reaction bythe presence of a protecting group. Those of skill in the art understandhow to protect a particular functional group such that it does notinterfere with a chosen set of reaction conditions. For examples ofuseful protecting groups, see, for example, Greene et al., PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.

In the discussion that follows, a number of specific examples ofmodified sugars that are useful in practicing the present invention areset forth. In the exemplary embodiments, a sialic acid derivative isutilized as the sugar nucleus to which the modifying group is attached.The focus of the discussion on sialic acid derivatives is for clarity ofillustration only and should not be construed to limit the scope of theinvention. Those of skill in the art will appreciate that a variety ofother sugar moieties can be activated and derivatized in a manneranalogous to that set forth using sialic acid as an example. Forexample, numerous methods are available for modifying galactose,glucose, N-acetylgalactosamine and fucose to name a few sugarsubstrates, which are readily modified by art recognized methods. See,for example, Elhalabi et al., Curr. Med. Chem. 6: 93 (1999); and Schaferet al., J. Org. Chem. 65: 24 (2000).

In an exemplary embodiment, the peptide that is modified by a method ofthe invention is a peptide that is produced in mammalian cells (e.g.,CHO cells) or in a transgenic animal and thus, contains N— and/orO-linked oligosaccharide chains, which are incompletely sialylated. Theoligosaccharide chains of the glycopeptide lacking a sialic acid andcontaining a terminal galactose residue can be PEGylated, PPGylated orotherwise modified with a modified sialic acid.

In Scheme 4, the mannosamine glycoside 1, is treated with the activeester of a protected amino acid (e.g., glycine) derivative, convertingthe sugar amine residue into the corresponding protected amino acidamide adduct. The adduct is treated with an aldolase to form the sialicacid 2. Compound 2 is converted to the corresponding CMP derivative bythe action of CMP-SA synthetase, followed by catalytic hydrogenation ofthe CMP derivative to produce compound 3. The amine introduced viaformation of the glycine adduct is utilized as a locus of PEG or PPGattachment by reacting compound 3 with an activated PEG or PPGderivative (e.g., PEG-C(O)NHS, PPG-C(O)NHS), producing 4 or 5,respectively.

Table 3 sets forth representative examples of sugar monophosphates thatare derivatized with a PEG or PPG moiety. Certain of the compounds ofTable 3 are prepared by the method of Scheme 1. Other derivatives areprepared by art-recognized methods. See, for example, Keppler et al.,Glycobiology 11: 11R (2001); and Charter et al., Glycobiology 10: 1049(2000)). Other amine reactive PEG and PPG analogues are commerciallyavailable, or they can be prepared by methods readily accessible tothose of skill in the art.

TABLE 3 Examples of sugar monophosphates that are derivatized with a PEGor PPG moiety

CMP-KDN-5-O-R

CMP-NeuAc-9-NH-R

CMP-NeuAc-8-O-R

CMP-NeuAc-8-NH-R

CMP-NeuAc-7-O-R

CMF-NeuAc-7-NH-R

CMP-NeuAc-4-O-R

CMP-NeuAc-4-NH-R

CMP-SA-5-NH-R

CMP-NeuAc-9-O-R

The modified sugar phosphates of use in practicing the present inventioncan be substituted in other positions as well as those set forth above.“i” may be Na or another salt and “i” may be interchangeable with Na.Presently preferred substitutions of sialic acid are set forth inFormula 5.

in which X is a linking group, which is preferably selected from —O—,—N(H)—, —S, CH₂—, and N(R)₂, in which each R is a member independentlyselected from R¹—R⁵. “i” may be Na or another salt, and Na may beinterchangeable with “i: The symbols Y, Z, A and B each represent agroup that is selected from the group set forth above for the identityof X. X, Y, Z, A and B are each independently selected and, therefore,they can be the same or different. The symbols R¹, R², R³, R⁴ and R⁵represent H, polymers, a water-soluble polymer, therapeutic moiety,biomolecule or other moiety. The symbol R6 represents H, OH, or apolymer. Alternatively, these symbols represent a linker that is linkedto a polymer, water-soluble polymer, therapeutic moiety, biomolecule orother moiety.

In another exemplary embodiment, a mannosamine is simultaneouslyacylated and activated for a nucleophilic substitution by the use ofchloroacetic anhydride as set forth in Scheme 5. In each of the schemespresented in this section, i⁺ or Na⁺ can be interchangeable, wherein thesalt can be sodium, or can be any other suitable salt.

The resulting chloro-derivatized glycan is contacted with pyruvate inthe presence of an aldolase, forming a chloro-derivatized sialic acid.The corresponding nucleotide sugar is prepared by contacted the sialicacid derivative with an appropriate nucleotide triphosphates and asynthetase. The chloro group on the sialic acid moiety is then displacedwith a nucleophilic PEG derivative, such as thio-PEG.

In a further exemplary embodiment, as shown is Scheme 6, a mannosamineis acylated with a bis-HOBT dicarboxylate, producing the correspondingamido-alkyl-carboxylic acid, which is subsequently converted to a sialicacid derivative. The sialic acid derivative is converted to a nucleotidesugar, and the carboxylic acid is activated and reacted with anucleophilic PEG derivative, such as amino-PEG.

In another exemplary embodiment, set forth in Scheme 7, amine- andcarboxyl-protected neuraminic acid is activated by converting theprimary hydroxyl group to the corresponding p-toluenesulfonate ester,and the methyl ester is cleaved. The activated neuraminic acid isconverted to the corresponding nucleotide sugar, and the activatinggroup is displaced by a nucleophilic PEG species, such as thio-PEG.

In yet a further exemplary embodiment, as set forth in Scheme 8, theprimary hydroxyl moiety of an amine- and carboxyl-protected neuraminicacid derivative is alkylated using an electrophilic PEG, such aschloro-PEG. The methyl ester is subsequently cleaved and the PEG-sugaris converted to a nucleotide sugar.

Glycans other than sialic acid can be derivatized with PEG using themethods set forth herein. The derivatized glycans, themselves, are alsowithin the scope of the invention. Thus, Scheme 9 provides an exemplarysynthetic route to a PEGylated galactose nucleotide sugar. The primaryhydroxyl group of galactose is activated as the correspondingtoluenesulfonate ester, which is subsequently converted to a nucleotidesugar.

Scheme 10 sets forth an exemplary route for preparing a galactose-PEGderivative that is based upon a galactose-6-amine moiety. Thus,galactosamine is converted to a nucleotide sugar, and the amine moietyof galactosamine is functionalized with an active PEG derivative.

Scheme 11 provides another exemplary route to galactose derivatives. Thestarting point for Scheme 11 is galactose-2-amine, which is converted toa nucleotide sugar. The amine moiety of the nucleotide sugar is thelocus for attaching a PEG derivative, such as Methoxy-PEG (mPEG)carboxylic acid.

Exemplary moieties attached to the conjugates disclosed herein include,but are not limited to, PEG derivatives (e.g., acyl-PEG, acyl-alkyl-PEG,alkyl-acyl-PEG carbamoyl-PEG, aryl-PEG, alkyl-PEG), PPG derivatives(e.g., acyl-PPG, acyl-alkyl-PPG, alkyl-acyl-PPG carbamoyl-PPG,aryl-PPG), polyapartic acid, polyglutamate, polylysine, therapeuticmoieties, diagnostic moieties, mannose-6-phosphate, heparin, heparan,SLe^(x), mannose, mannose-6-phosphate, Sialyl Lewis X, FGF, VFGF,proteins (e.g., transferrin), chondroitin, keratan, dermatan, dextran,modified dextran, amylose, bisphosphate, poly-SA, hyaluronic acid,keritan, albumin, integrins, antennary oligosaccharides, peptides andthe like. Methods of conjugating the various modifying groups to asaccharide moiety are readily accessible to those of skill in the art(POLY(ETHYLENE GLYCOL CHEMISTRY: BIOTECHNCAL AND BIOMEDICALAPPLICATIONS, J. Milton Harris, Ed., Plenum Pub. Corp., 1992; POLY(ETHYLENE GLYCOL) CHEMICAL AND BIOLOGICAL APPLICATIONS, J. MiltonHarris, Ed., ACS Symposium Series No. 680, American Chemical Society,1997; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego,1996; and Dunn et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS,ACS Symposium Series Vol. 469, American Chemical Society, Washington,D.C. 1991).

Purification of Sugars, Nucleotide Sugars and Derivatives

The nucleotide sugars and derivatives produced by the above processescan be used without purification. However, it is usually preferred torecover the product. Standard, well-known techniques for recovery ofglycosylated saccharides such as thin or thick layer chromatography,column chromatography, ion exchange chromatography, or membranefiltration can be used. It is preferred to use membrane filtration, morepreferably utilizing a reverse osmotic membrane, or one or more columnchromatographic techniques for the recovery as is discussed hereinafterand in the literature cited herein. For instance, membrane filtrationwherein the membranes have molecular weight cutoff of about 3000 toabout 10,000 can be used to remove proteins for reagents having amolecular weight of less than 10,000 Da. Membrane filtration or reverseosmosis can then be used to remove salts and/or purify the productsaccharides (see, e.g., WO 98/15581). Nanofilter membranes are a classof reverse osmosis membranes that pass monovalent salts but retainpolyvalent salts and uncharged solutes larger than about 100 to about2,000 Daltons, depending upon the membrane used. Thus, in a typicalapplication, saccharides prepared by the methods of the presentinvention will be retained in the membrane and contaminating salts willpass through.

G. Cross-Linking Groups

Preparation of the modified sugar for use in the methods of the presentinvention includes attachment of a modifying group to a sugar residueand forming a stable adduct, which is a substrate for aglycosyltransferase. Thus, it is often preferred to use a cross-linkingagent to conjugate the modifying group and the sugar. Exemplarybifunctional compounds which can be used for attaching modifying groupsto carbohydrate moieties include, but are not limited to, bifunctionalpoly(ethylene glycols), polyamides, polyethers, polyesters and the like.General approaches for linking carbohydrates to other molecules areknown in the literature. See, for example, Lee et al., Biochemistry 28:1856 (1989); Bhatia et al., Anal. Biochem. 178: 408 (1989); Janda etal., J. Am. Chem. Soc. 112: 8886 (1990) and Bednarski et al., WO92/18135. In the discussion that follows, the reactive groups aretreated as benign on the sugar moiety of the nascent modified sugar. Thefocus of the discussion is for clarity of illustration. Those of skillin the art will appreciate that the discussion is relevant to reactivegroups on the modifying group as well.

An exemplary strategy involves incorporation of a protected sulfhydrylonto the sugar using the heterobifunctional crosslinker SPDP(n-succinimidyl-3-(2-pyridyldithio)propionate and then deprotecting thesulfhydryl for formation of a disulfide bond with another sulfhydryl onthe modifying group.

If SPDP detrimentally affects the ability of the modified sugar to actas a glycosyltransferase substrate, one of an array of othercrosslinkers such as 2-iminothiolane or N-succinimidylS-acetylthioacetate (SATA) is used to form a disulfide bond.2-iminothiolane reacts with primary amines, instantly incorporating anunprotected sulfhydryl onto the amine-containing molecule. SATA alsoreacts with primary amines, but incorporates a protected sulfhydryl,which is later deacetylated using hydroxylamine to produce a freesulfhydryl. In each case, the incorporated sulfhydryl is free to reactwith other sulfhydryls or protected sulfhydryl, like SPDP, forming therequired disulfide bond.

The above-described strategy is exemplary, and not limiting, of linkersof use in the invention. Other crosslinkers are available that can beused in different strategies for crosslinking the modifying group to thepeptide. For example, TPCH(S-(2-thiopyridyl)-L-cysteine hydrazide andTPMPH ((S-(2-thiopyridyl) mercapto-propionohydrazide) react withcarbohydrate moieties that have been previously oxidized by mildperiodate treatment, thus forming a hydrazone bond between the hydrazideportion of the crosslinker and the periodate generated aldehydes. TPCHand TPMPH introduce a 2-pyridylthione protected sulfhydryl group ontothe sugar, which can be deprotected with DTT and then subsequently usedfor conjugation, such as forming disulfide bonds between components.

If disulfide bonding is found unsuitable for producing stable modifiedsugars, other crosslinkers may be used that incorporate more stablebonds between components. The heterobifunctional crosslinkers GMBS(N-gama-malimidobutyryloxy)succinimide) and SMCC (succinimidyl4-(N-maleimido-methyl)cyclohexane) react with primary amines, thusintroducing a maleimide group onto the component. The maleimide groupcan subsequently react with sulfhydryls on the other component, whichcan be introduced by previously mentioned crosslinkers, thus forming astable thioether bond between the components. If steric hindrancebetween components interferes with either component's activity or theability of the modified sugar to act as a glycosyltransferase substrate,crosslinkers can be used which introduce long spacer arms betweencomponents and include derivatives of some of the previously mentionedcrosslinkers (i.e., SPDP). Thus, there is an abundance of suitablecrosslinkers, which are useful; each of which is selected depending onthe effects it has on optimal peptide conjugate and modified sugarproduction.

A variety of reagents are used to modify the components of the modifiedsugar with intramolecular chemical crosslinks (for reviews ofcrosslinking reagents and crosslinking procedures see: Wold, F., Meth.Enzymol. 25: 623-651, 1972; Weetall, H. H., and Cooney, D. A., In:ENZYMES AS DRUGS. (Holcenberg, and Roberts, eds.) pp. 395-442, Wiley,New York, 1981; Ji, T. H., Meth. Enzymol. 91: 580-609, 1983; Mattson etal., Mol. Biol. Rep. 17: 167-183, 1993, all of which are incorporatedherein by reference). Preferred crosslinking reagents are derived fromvarious zero-length, homo-bifunctional, and hetero-bifunctionalcrosslinking reagents. Zero-length crosslinking reagents include directconjugation of two intrinsic chemical groups with no introduction ofextrinsic material. Agents that catalyze formation of a disulfide bondbelong to this category. Another example is reagents that inducecondensation of a carboxyl and a primary amino group to form an amidebond such as carbodiimides, ethylchloroformate, Woodward's reagent K(2-ethyl-5-phenylisoxazolium-3′-sulfonate), and carbonyldiimidazole. Inaddition to these chemical reagents, the enzyme transglutaminase(glutamyl-peptide γ-glutamyltransferase; EC 2.3.2.13) may be used aszero-length crosslinking reagent. This enzyme catalyzes acyl transferreactions at carboxamide groups of protein-linked glutaminyl residues,usually with a primary amino group as substrate. Preferred homo- andhetero-bifunctional reagents contain two identical or two dissimilarsites, respectively, which may be reactive for amino, sulfhydryl,guanidino, indole, or nonspecific groups.

2. Preferred Specific Sites in Crosslinking Reagents

a. Amino-Reactive Groups

In one preferred embodiment, the sites on the cross-linker areamino-reactive groups. Useful non-limiting examples of amino-reactivegroups include N-hydroxysuccinimide (NHS) esters, imidoesters,isocyanates, acylhalides, arylazides, p-nitrophenyl esters, aldehydes,and sulfonyl chlorides.

NHS esters react preferentially with the primary (including aromatic)amino groups of a modified sugar component. The imidazole groups ofhistidines are known to compete with primary amines for reaction, butthe reaction products are unstable and readily hydrolyzed. The reactioninvolves the nucleophilic attack of an amine on the acid carboxyl of anNHS ester to form an amide, releasing the N-hydroxysuccinimide. Thus,the positive charge of the original amino group is lost.

Imidoesters are the most specific acylating reagents for reaction withthe amine groups of the modified sugar components. At a pH between 7 and10, imidoesters react only with primary amines. Primary amines attackimidates nucleophilically to produce an intermediate that breaks down toamidine at high pH or to a new imidate at low pH. The new imidate canreact with another primary amine, thus crosslinking two amino groups, acase of a putatively monofunctional imidate reacting bifunctionally. Theprincipal product of reaction with primary amines is an amidine that isa stronger base than the original amine. The positive charge of theoriginal amino group is therefore retained.

Isocyanates (and isothiocyanates) react with the primary amines of themodified sugar components to form stable bonds. Their reactions withsulfhydryl, imidazole, and tyrosyl groups give relatively unstableproducts.

Acylazides are also used as amino-specific reagents in whichnucleophilic amines of the affinity component attack acidic carboxylgroups under slightly alkaline conditions, e.g. pH 8.5.

Arylhalides such as 1,5-difluoro-2,4-dinitrobenzene react preferentiallywith the amino groups and tyrosine phenolic groups of modified sugarcomponents, but also with sulfhydryl and imidazole groups.

p-Nitrophenyl esters of mono- and dicarboxylic acids are also usefulamino-reactive groups. Although the reagent specificity is not veryhigh, α- and ε-amino groups appear to react most rapidly.

Aldehydes such as glutaraldehyde react with primary amines of modifiedsugar. Although unstable Schiff bases are formed upon reaction of theamino groups with the aldehydes of the aldehydes, glutaraldehyde iscapable of modifying the modified sugar with stable crosslinks. At pH6-8, the pH of typical crosslinking conditions, the cyclic polymersundergo a dehydration to form α-β unsaturated aldehyde polymers. Schiffbases, however, are stable, when conjugated to another double bond. Theresonant interaction of both double bonds prevents hydrolysis of theSchiff linkage. Furthermore, amines at high local concentrations canattack the ethylenic double bond to form a stable Michael additionproduct.

Aromatic sulfonyl chlorides react with a variety of sites of themodified sugar components, but reaction with the amino groups is themost important, resulting in a stable sulfonamide linkage.

b. Sulfhydryl-Reactive Groups

In another preferred embodiment, the sites are sulfhydryl-reactivegroups. Useful, non-limiting examples of sulfhydryl-reactive groupsinclude maleimides, alkyl halides, pyridyl disulfides, andthiophthalimides.

Maleimides react preferentially with the sulfhydryl group of themodified sugar components to form stable thioether bonds. They alsoreact at a much slower rate with primary amino groups and the imidazolegroups of histidines. However, at pH 7 the maleimide group can beconsidered a sulfhydryl-specific group, since at this pH the reactionrate of simple thiols is 1000-fold greater than that of thecorresponding amine.

Alkyl halides react with sulfhydryl groups, sulfides, imidazoles, andamino groups. At neutral to slightly alkaline pH, however, alkyl halidesreact primarily with sulfhydryl groups to form stable thioether bonds.At higher pH, reaction with amino groups is favored.

Pyridyl disulfides react with free sulfhydryls via disulfide exchange togive mixed disulfides. As a result, pyridyl disulfides are the mostspecific sulfhydryl-reactive groups.

Thiophthalimides react with free sulfhydryl groups to form disulfides.

c. Carboxyl-Reactive Residue

In another embodiment, carbodiimides soluble in both water and organicsolvent, are used as carboxyl-reactive reagents. These compounds reactwith free carboxyl groups forming a pseudourea that can then coupled toavailable amines yielding an amide linkage. Procedures to modify acarboxyl group with carbodiimide is well know in the art (see, Yamada etal. Biochemistry 20: 4836-4842, 1981).

3. Preferred Nonspecific Sites in Crosslinking Reagents

In addition to the use of site-specific reactive moieties, the presentinvention contemplates the use of non-specific reactive groups to linkthe sugar to the modifying group.

Exemplary non-specific cross-linkers include photoactivatable groups,completely inert in the dark, which are converted to reactive speciesupon absorption of a photon of appropriate energy. In one preferredembodiment, photoactivatable groups are selected from precursors ofnitrenes generated upon heating or photolysis of azides.Electron-deficient nitrenes are extremely reactive and can react with avariety of chemical bonds including N—H, O—H, C—H, and C═C. Althoughthree types of azides (aryl, alkyl, and acyl derivatives) may beemployed, arylazides are presently preferred. The reactivity ofarylazides upon photolysis is better with N—H and O—H than C—H bonds.Electron-deficient arylnitrenes rapidly ring-expand to formdehydroazepines, which tend to react with nucleophiles, rather than formC—H insertion products. The reactivity of arylazides can be increased bythe presence of electron-withdrawing substituents such as nitro orhydroxyl groups in the ring. Such substituents push the absorptionmaximum of arylazides to longer wavelength. Unsubstituted arylazideshave an absorption maximum in the range of 260-280 nm, while hydroxy andnitroarylazides absorb significant light beyond 305 nm. Therefore,hydroxy and nitroarylazides are most preferable since they allow toemploy less harmful photolysis conditions for the affinity componentthan unsubstituted arylazides.

In another preferred embodiment, photoactivatable groups are selectedfrom fluorinated arylazides. The photolysis products of fluorinatedarylazides are arylnitrenes, all of which undergo the characteristicreactions of this group, including C—H bond insertion, with highefficiency (Keana et al., J. Org. Chem. 55: 3640-3647, 1990).

In another embodiment, photoactivatable groups are selected frombenzophenone residues. Benzophenone reagents generally give highercrosslinking yields than arylazide reagents.

In another embodiment, photoactivatable groups are selected from diazocompounds, which form an electron-deficient carbene upon photolysis.These carbenes undergo a variety of reactions including insertion intoC—H bonds, addition to double bonds (including aromatic systems),hydrogen attraction and coordination to nucleophilic centers to givecarbon ions.

In still another embodiment, photoactivatable groups are selected fromdiazopyruvates. For example, the p-nitrophenyl ester of p-nitrophenyldiazopyruvate reacts with aliphatic amines to give diazopyruvic acidamides that undergo ultraviolet photolysis to form aldehydes. Thephotolyzed diazopyruvate-modified affinity component will react likeformaldehyde or glutaraldehyde forming crosslinks.

4. Homobifunctional Reagents

a. Homobifunctional Crosslinkers Reactive With Primary Amines

Synthesis, properties, and applications of amine-reactive cross-linkersare commercially described in the literature (for reviews ofcrosslinking procedures and reagents, see above). Many reagents areavailable (e.g., Pierce Chemical Company, Rockford, Ill.; Sigma ChemicalCompany, St. Louis, Mo.; Molecular Probes, Inc., Eugene, Oreg.).

Preferred, non-limiting examples of homobifunctional NHS esters includedisuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartarate (DST),disulfosuccinimidyl tartarate (sulfo-DST),bis-2-(succinimidooxycarbonyloxy)ethylsulfone (BSOCOES),bis-2-(sulfosuccinimidooxy-carbonyloxy)ethylsulfone (sulfo-BSOCOES),ethylene glycolbis(succinimidylsuccinate) (EGS), ethyleneglycolbis(sulfosuccinimidylsuccinate) (sulfo-EGS),dithiobis(succinimidyl-propionate (DSP), anddithiobis(sulfosuccinimidylpropionate (sulfo-DSP). Preferred,non-limiting examples of homobifunctional imidoesters include dimethylmalonimidate (DMM), dimethyl succinimidate (DMSC), dimethyl adipimidate(DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS),dimethyl-3,3′-oxydipropionimidate (DODP),dimethyl-3,3′-(methylenedioxy)dipropionimidate (DMDP),dimethyl-,3′-(dimethylenedioxy)dipropionimidate (DDDP),dimethyl-3,3′-(tetramethylenedioxy)-dipropionimidate (DTDP), anddimethyl-3,3′-dithiobispropionimidate (DTBP).

Preferred, non-limiting examples of homobifunctional isothiocyanatesinclude: p-phenylenediisothiocyanate (DITC), and4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS).

Preferred, non-limiting examples of homobifunctional isocyanates includexylene-diisocyanate, toluene-2,4-diisocyanate,toluene-2-isocyanate-4-isothiocyanate,3-methoxydiphenylmethane-4,4′-diisocyanate,2,2′-dicarboxy-4,4′-azophenyldiisocyanate, andhexamethylenediisocyanate.

Preferred, non-limiting examples of homobifunctional arylhalides include1,5-difluoro-2,4-dinitrobenzene (DFDNB), and4,4′-difluoro-3,3′-dinitrophenyl-sulfone.

Preferred, non-limiting examples of homobifunctional aliphatic aldehydereagents include glyoxal, malondialdehyde, and glutaraldehyde.

Preferred, non-limiting examples of homobifunctional acylating reagentsinclude nitrophenyl esters of dicarboxylic acids.

Preferred, non-limiting examples of homobifunctional aromatic sulfonylchlorides include phenol-2,4-disulfonyl chloride, andα-naphthol-2,4-disulfonyl chloride.

Preferred, non-limiting examples of additional amino-reactivehomobifunctional reagents include erythritolbiscarbonate which reactswith amines to give biscarbamates.

b. Homobifunctional Crosslinkers Reactive with Free Sulfhydryl Groups

Synthesis, properties, and applications of such reagents are describedin the literature (for reviews of crosslinking procedures and reagents,see above). Many of the reagents are commercially available (e.g.,Pierce Chemical Company, Rockford, Ill.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, Oreg.).

Preferred, non-limiting examples of homobifunctional maleimides includebismaleimidohexane (BMH), N,N′-(1,3-phenylene) bismaleimide,N,N′-(1,2-phenylene)bismaleimide, azophenyldimaleimide, andbis(N-maleimidomethyl)ether.

Preferred, non-limiting examples of homobifunctional pyridyl disulfidesinclude 1,4-di-3′-(2′-pyridyldithio)propionamidobutane (DPDPB).

Preferred, non-limiting examples of homobifunctional alkyl halidesinclude 2,2′-dicarboxy-4,4′-diiodoacetamidoazobenzene,α,α′-diiodo-p-xylenesulfonic acid, α,α′-dibromo-p-xylenesulfonic acid,N,N-bis(b-bromoacetyl)benzylamine, N,N′-di(bromoacetyl)phenylthydrazine,and 1,2-di(bromoacetyl)amino-3-phenylpropane.

c. Homobifunctional Photoactivatable Crosslinkers

Synthesis, properties, and applications of such reagents are describedin the literature (for reviews of crosslinking procedures and reagents,see above). Some of the reagents are commercially available (e.g.,Pierce Chemical Company, Rockford, Ill.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, Oreg.).

Preferred, non-limiting examples of homobifunctional photoactivatablecrosslinker include bis-β-(4-azidosalicylamido)ethyldisulfide (BASED),di-N-(2-nitro-4-azidophenyl)-cystamine-S,S-dioxide (DNCO), and4,4′-dithiobisphenylazide.

5. HeteroBifunctional Reagents

a. Amino-Reactive HeteroBifunctional Reagents With a Pyridyl DisulfideMoiety

Synthesis, properties, and applications of such reagents are describedin the literature (for reviews of crosslinking procedures and reagents,see above). Many of the reagents are commercially available (e.g.,Pierce Chemical Company, Rockford, Ill.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, Oreg.).

Preferred, non-limiting examples of hetero-bifunctional reagents with apyridyl disulfide moiety and an amino-reactive NHS ester includeN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl6-3-(2-pyridyldithio)propionamidohexanoate (LC-SPDP), sulfosuccinimidyl6-3-(2-pyridyldithio)propionamidohexanoate (sulfo-LCSPDP),4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT),and sulfosuccinimidyl 6-α-methyl-α-(2-pyridyldithio)toluamidohexanoate(sulfo-LC-SNTT).

b. Amino-Reactive HeteroBifunctional Reagents With a Maleimide Moiety

Synthesis, properties, and applications of such reagents are describedin the literature. Preferred, non-limiting examples ofhetero-bifunctional reagents with a maleimide moiety and anamino-reactive NHS ester include succinimidyl maleimidylacetate (AMAS),succinimidyl 3-maleimidylpropionate (BMPS),N-γ-maleimidobutyryloxysuccinimide ester(GMBS)N-γ-maleimidobutrryloxysulfo succinimide ester (sulfo-GMBS)succinimidyl 6-maleimidylhexanoate (EMCS), succinimidyl3-maleimidylbenzoate (SMB), m-maleimidobenzoyl-N-hydroxysuccinimideester (MBS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester(sulfo-MBS), succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC),sulfosuccinimidyl 4-(-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), andsulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB).

c. Amino-Reactive HeteroBifunctional Reagents With an Alkyl HalideMoiety

Synthesis, properties, and applications of such reagents are describedin the literature. Preferred, non-limiting examples ofhetero-bifunctional reagents with an alkyl halide moiety and anamino-reactive NHS ester includeN-succinimidyl-(4-iodoacetyl)arninobenzoate (SIAB),sulfosuccinimidyl-(4-iodoacetyl)aminobenzoate (sulfo-SIAB),succinimidyl-6-(iodoacetyl)aminohexanoate (SIAX),succinimidyl-6-(6-((iodoacetyl)-amino)hexanoylamino)hexanoate (SIAXX,succinimidyl-6-(((4-(iodoacetyl)-amino)-methyl)-cyclohexane-1-carbonyl)aminohexanoate(SIACX), andsuccinimidyl-4((iodoacetyl)-amino)methylcyclohexane-1-carboxylate(SIAC).

A preferred example of a hetero-bifunctional reagent with anamino-reactive NHS ester and an alkyl dihalide moiety isN-hydroxysuccinimidyl 2,3-dibromopropionate (SDBP). SDBP introducesintramolecular crosslinks to the affinity component by conjugating itsamino groups. The reactivity of the dibromopropionyl moiety towardsprimary amine groups is controlled by the reaction temperature (McKenzieet al., Protein Chem. 7: 581-592 (1988)).

Preferred, non-limiting examples of hetero-bifunctional reagents with analkyl halide moiety and an amino-reactive p-nitrophenyl ester moietyinclude p-nitrophenyl iodoacetate (NPIA).

Other cross-linking agents are known to those of skill in the art. See,for example, Pomato et al., U.S. Pat. No. 5,965,106. It is within theabilities of one of skill in the art to choose an appropriatecross-linking agent for a particular application.

d. Cleavable Linker Groups

In yet a further embodiment, the linker group is provided with a groupthat can be cleaved to release the modifying group from the sugarresidue. Many cleavable groups are known in the art. See, for example,Jung et al., Biochem. Biophys. Acta 761: 152-162 (1983); Joshi et al.,J. Biol. Chem. 265: 14518-14525 (1990); Zarling et al., J. Immunol. 124:913-920 (1980); Bouizar et al., Eur. J. Biochem. 155: 141-147 (1986);Park et al., J. Biol. Chem. 261: 205-210 (1986); Browning et al., J.Immunol. 143: 1859-1867 (1989). Moreover a broad range of cleavable,bifunctional (both homo- and hetero-bifunctional) linker groups iscommercially available from suppliers such as Pierce.

Exemplary cleavable moieties can be cleaved using light, heat orreagents such as thiols, hydroxylamine, bases, periodate and the like.Moreover, certain preferred groups are cleaved in vivo in response tobeing endocytosed (e.g., cis-aconityl; see, Shen et al., Biochem.Biophys. Res. Commun. 102: 1048 (1991)). Preferred cleavable groupscomprise a cleavable moiety which is a member selected from the groupconsisting of disulfide, ester, imide, carbonate, nitrobenzyl, phenacyland benzoin groups.

e. Conjugation of Modified Sugars to Peptides

The modified sugars are conjugated to a glycosylated or non-glycosylatedpeptide using an appropriate enzyme to mediate the conjugation.Preferably, the concentrations of the modified donor sugar(s), enzyme(s)and acceptor peptide(s) are selected such that glycosylation proceedsuntil the acceptor is consumed. The considerations discussed below,while set forth in the context of a sialyltransferase, are generallyapplicable to other glycosyltransferase reactions.

A number of methods of using glycosyltransferases to synthesize desiredoligosaccharide structures are known and are generally applicable to theinstant invention. Exemplary methods are described, for instance, WO96/32491, Ito et al., Pure Appl. Chem. 65: 753 (1993), and U.S. Pat.Nos. 5,352,670, 5,374,541, and 5,545,553.

The present invention is practiced using a single glycosyltransferase ora combination of glycosyltransferases. For example, one can use acombination of a sialyltransferase and a galactosyltransferase. In thoseembodiments using more than one enzyme, the enzymes and substrates arepreferably combined in an initial reaction mixture, or the enzymes andreagents for a second enzymatic reaction are added to the reactionmedium once the first enzymatic reaction is complete or nearly complete.By conducting two enzymatic reactions in sequence in a single vessel,overall yields are improved over procedures in which an intermediatespecies is isolated. Moreover, cleanup and disposal of extra solventsand by-products is reduced.

In a preferred embodiment, each of the first and second enzyme is aglycosyltransferase. In another preferred embodiment, one enzyme is anendoglycosidase. In another preferred embodiment, one enzyme is anexoglycosidase. In an additional preferred embodiment, more than twoenzymes are used to assemble the modified glycoprotein of the invention.The enzymes are used to alter a saccharide structure on the peptide atany point either before or after the addition of the modified sugar tothe peptide.

In another embodiment, at least two of the enzymes areglycosyltransferases and the last sugar added to the saccharidestructure of the peptide is a non-modified sugar. Instead, the modifiedsugar is internal to the glycan structure and therefore need not be theultimate sugar on the glycan. In an exemplary embodiment,galactosyltransferase may catalyze the transfer of Gal-PEG fromUDP-Gal-PEG onto the glycan, followed by incubation in the presence ofST3Gal3 and CMP-SA, which serves to add a “capping” unmodified sialicacid onto the glycan (FIG. 23A).

In another embodiment, at least two of the enzymes used areglycosyltransferases, and at least two modified sugars are added to theglycan structures on the peptide. In this manner, two or more differentglycoconjugates may be added to one or more glycans on a peptide. Thisprocess generates glycan structures having two or more functionallydifferent modified sugars. In an exemplary embodiment, incubation of thepeptide with GnT-I, II and UDP-GlcNAc-PEG serves to add a GlcNAc-PEGmolecule to the glycan; incubation with galactosyltransferase andUDP-Gal then serves to add a Gal residue thereto; and, incubation withST3Gal3 and CMP-SA-Man-6-Phosphate serves to add aSA-mannose-6-phosphate molecule to the glycan. This series of reactionsresults in a glycan chain having the functional characteristics of aPEGylated glycan as well as mannose-6-phosphate targeting activity (FIG.23B).

In another embodiment, at least two of the enzymes used in the reactionare glycosyltransferases, and again, different modified sugars are addedto N-linked and O-linked glycans on the peptide. This embodiment isuseful when two different modified sugars are to be added to the glycansof a peptide, but when it is important to spatially separate themodified sugars on the peptide from each other. For example, if themodified sugars comprise bulky molecules, including but not limited to,PEG and other molecules such as a linker molecule, this method may bepreferable. The modified sugars may be added simultaneously to theglycan structures on a peptide, or they may be added sequentially. In anexemplary embodiment, incubation with ST3Gal3 and CMP-SA-PEG serves toadd sialic acid-PEG to the N-linked glycans, while incubation withST3Gal1 and CMP-SA-bisPhosphonate serves to add sialicacid-BisPhosphonate to the O-linked glycans (FIG. 23C).

In another embodiment, the method makes use of one or more exo- orendoglycosidase. The glycosidase is typically a mutant, which isengineered to form glycosyl bonds rather than rupture them. The mutantglycanase, sometimes called a glycosynthase, typically includes asubstitution of an amino acid residue for an active site acidic aminoacid residue. For example, when the endoglycanase is endo-H, thesubstituted active site residues will typically be Asp at position 130,Glu at position 132 or a combination thereof. The amino acids aregenerally replaced with serine, alanine, asparagine, or glutamine.Exoglycosidases such as transialylidase are also useful.

The mutant enzyme catalyzes the reaction, usually by a synthesis stepthat is analogous to the reverse reaction of the endoglycanasehydrolysis step. In these embodiments, the glycosyl donor molecule(e.g., a desired oligo- or mono-saccharide structure) contains a leavinggroup and the reaction proceeds with the addition of the donor moleculeto a GlcNAc residue on the protein. For example, the leaving group canbe a halogen, such as fluoride. In other embodiments, the leaving groupis a Asn, or a Asn-peptide moiety. In yet further embodiments, theGlcNAc residue on the glycosyl donor molecule is modified. For example,the GlcNAc residue may comprise a 1,2 oxazoline moiety.

In a preferred embodiment, each of the enzymes utilized to produce aconjugate of the invention are present in a catalytic amount. Thecatalytic amount of a particular enzyme varies according to theconcentration of that enzyme's substrate as well as to reactionconditions such as temperature, time and pH value. Means for determiningthe catalytic amount for a given enzyme under preselected substrateconcentrations and reaction conditions are well known to those of skillin the art.

The temperature at which an above-described process is carried out canrange from just above freezing to the temperature at which the mostsensitive enzyme denatures. Preferred temperature ranges are about 0° C.to about 55° C., and more preferably about 20° C. to about 37° C. Inanother exemplary embodiment, one or more components of the presentmethod are conducted at an elevated temperature using a thermophilicenzyme.

The reaction mixture is maintained for a period of time sufficient forthe acceptor to be glycosylated, thereby forming the desired conjugate.Some of the conjugate can often be detected after a few hours, withrecoverable amounts usually being obtained within 24 hours or less.Those of skill in the art understand that the rate of reaction isdependent on a number of variable factors (e.g., enzyme concentration,donor concentration, acceptor concentration, temperature, solventvolume), which are optimized for a selected system.

The present invention also provides for the industrial-scale productionof modified peptides. As used herein, an industrial scale generallyproduces at least one gram of finished, purified conjugate.

In the discussion that follows, the invention is exemplified by theconjugation of modified sialic acid moieties to a glycosylated peptide.The exemplary modified sialic acid is labeled with PEG. The focus of thefollowing discussion on the use of PEG-modified sialic acid andglycosylated peptides is for clarity of illustration and is not intendedto imply that the invention is limited to the conjugation of these twopartners. One of skill understands that the discussion is generallyapplicable to the additions of modified glycosyl moieties other thansialic acid. Moreover, the discussion is equally applicable to themodification of a glycosyl unit with agents other than PEG includingother water-soluble polymers, therapeutic moieties, and biomolecules.

An enzymatic approach can be used for the selective introduction ofPEGylated or PPGylated carbohydrates onto a peptide or glycopeptide. Themethod utilizes modified sugars containing PEG, PPG, or a maskedreactive functional group, and is combined with the appropriateglycosyltransferase or glycosynthase. By selecting theglycosyltransferase that will make the desired carbohydrate linkage andutilizing the modified sugar as the donor substrate, the PEG or PPG canbe introduced directly onto the peptide backbone, onto existing sugarresidues of a glycopeptide or onto sugar residues that have been addedto a peptide.

An acceptor for the sialyltransferase is present on the peptide to bemodified by the methods of the present invention either as a naturallyoccurring structure or one placed there recombinantly, enzymatically orchemically. Suitable acceptors, include, for example, galactosylacceptors such as Galβ1, 4GlcNAc, Galβ1, 4GalNAc, Galβ1, 3GalNAc,lacto-N-tetraose, Galβ1, 3GlcNAc, Galβ1, 3Ara, Galβ1, 6GlcNAc, Galβ1,4Glc (lactose), and other acceptors known to those of skill in the art(see, e.g., Paulson et al., J. Biol. Chem. 253: 5617-5624 (1978)).

In one embodiment, an acceptor for the sialyltransferase is present onthe peptide to be modified upon in vivo synthesis of the peptide. Suchpeptides can be sialylated using the claimed methods without priormodification of the glycosylation pattern of the peptide. Alternatively,the methods of the invention can be used to sialylate a peptide thatdoes not include a suitable acceptor; one first modifies the peptide toinclude an acceptor by methods known to those of skill in the art. In anexemplary embodiment, a GalNAc residue is added by the action of aGalNAc transferase.

In an exemplary embodiment, the galactosyl acceptor is assembled byattaching a galactose residue to an appropriate acceptor linked to thepeptide, e.g., a GlcNAc. The method includes incubating the peptide tobe modified with a reaction mixture that contains a suitable amount of agalactosyltransferase (e.g., galβ1,3 or galβ1,4), and a suitablegalactosyl donor (e.g., UDP-galactose). The reaction is allowed toproceed substantially to completion or, alternatively, the reaction isterminated when a preselected amount of the galactose residue is added.Other methods of assembling a selected saccharide acceptor will beapparent to those of skill in the art.

In yet another embodiment, peptide-linked oligosaccharides are first“trimmed,” either in whole or in part, to expose either an acceptor forthe sialyltransferase or a moiety to which one or more appropriateresidues can be added to obtain a suitable acceptor. Enzymes such asglycosyltransferases and endoglycosidases (see, for example U.S. Pat.No. 5,716,812) are useful for the attaching and trimming reactions. Adetailed discussion of “trimming” and remodeling N-linked and O-linkedglycans is provided elsewhere herein.

In the discussion that follows, the method of the invention isexemplified by the use of modified sugars having a water-soluble polymerattached thereto. The focus of the discussion is for clarity ofillustration. Those of skill will appreciate that the discussion isequally relevant to those embodiments in which the modified sugar bearsa therapeutic moiety, biomolecule or the like.

An exemplary embodiment of the invention in which a carbohydrate residueis “trimmed” prior to the addition of the modified sugar is set forth inFIG. 14, which sets forth a scheme in which high mannose is trimmed backto the first generation biantennary structure. A modified sugar bearinga water-soluble polymer is conjugated to one or more of the sugarresidues exposed by the “trimming back.” In one example, a water-solublepolymer is added via a GlcNAc moiety conjugated to the water-solublepolymer. The modified GlcNAc is attached to one or both of the terminalmannose residues of the biantennary structure. Alternatively, anunmodified GlcNAc can be added to one or both of the termini of thebranched species.

In another exemplary embodiment, a water-soluble polymer is added to oneor both of the terminal mannose residues of the biantennary structurevia a modified sugar having a galactose residue, which is conjugated toa GlcNAc residue added onto the terminal mannose residues.Alternatively, an unmodified Gal can be added to one or both terminalGlcNAc residues.

In yet a further example, a water-soluble polymer is added onto a Galresidue using a modified sialic acid.

Another exemplary embodiment is set forth in FIG. 15, which displays ascheme similar to that shown in FIG. 14, in which the high mannosestructure is “trimmed back” to the mannose from which the biantennarystructure branches. In one example, a water-soluble polymer is added viaa GlcNAc modified with the polymer. Alternatively, an unmodified GlcNAcis added to the mannose, followed by a Gal with an attachedwater-soluble polymer. In yet another embodiment, unmodified GlcNAc andGal residues are sequentially added to the mannose, followed by a sialicacid moiety modified with a water-soluble polymer.

FIG. 16 sets forth a further exemplary embodiment using a scheme similarto that shown in FIG. 14, in which high mannose is “trimmed back” to theGlcNAc to which the first mannose is attached. The GlcNAc is conjugatedto a Gal residue bearing a water-soluble polymer. Alternatively, anunmodified Gal is added to the GlcNAc, followed by the addition of asialic acid modified with a water-soluble sugar. In yet a furtherexample, the terminal GlcNAc is conjugated with Gal and the GlcNAc issubsequently fucosylated with a modified fucose bearing a water-solublepolymer.

FIG. 17 is a scheme similar to that shown in FIG. 14, in which highmannose is trimmed back to the first GlcNAc attached to the Asn of thepeptide. In one example, the GlcNAc of the GlcNAc-(Fuc)_(a) residue isconjugated with a GlcNAc bearing a water soluble polymer. In anotherexample, the GlcNAc of the GlcNAc-(Fuc)_(a) residue is modified withGal, which bears a water soluble polymer. In a still further embodiment,the GlcNAc is modified with Gal, followed by conjugation to the Gal of asialic acid modified with a water-soluble polymer.

Other exemplary embodiments are set forth in FIGS. 18-22. Anillustration of the array of reaction types with which the presentinvention may be practiced is provided in each of the aforementionedfigures.

The Examples set forth above provide an illustration of the power of themethods set forth herein. Using the methods of the invention, it ispossible to “trim back” and build up a carbohydrate residue ofsubstantially any desired structure. The modified sugar can be added tothe termini of the carbohydrate moiety as set forth above, or it can beintermediate between the peptide core and the terminus of thecarbohydrate.

In an exemplary embodiment, an existing sialic acid is removed from aglycopeptide using a sialidase, thereby unmasking all or most of theunderlying galactosyl residues. Alternatively, a peptide or glycopeptideis labeled with galactose residues, or an oligosaccharide residue thatterminates in a galactose unit. Following the exposure of or addition ofthe galactose residues, an appropriate sialyltransferase is used to adda modified sialic acid. The approach is summarized in Scheme 12.

In yet a further approach, summarized in Scheme 13, a masked reactivefunctionality is present on the sialic acid. The masked reactive groupis preferably unaffected by the conditions used to attach the modifiedsialic acid to the peptide. After the covalent attachment of themodified sialic acid to the peptide, the mask is removed and the peptideis conjugated with an agent such as PEG, PPG, a therapeutic moiety,biomolecule or other agent. The agent is conjugated to the peptide in aspecific manner by its reaction with the unmasked reactive group on themodified sugar residue.

Any modified sugar can be used with its appropriate glycosyltransferase,depending on the terminal sugars of the oligosaccharide side chains ofthe glycopeptide (Table 4). As discussed above, the terminal sugar ofthe glycopeptide required for introduction of the PEGylated or PPGylatedstructure can be introduced naturally during expression or it can beproduced post expression using the appropriate glycosidase(s),glycosyltransferase(s) or mix of glycosidase(s) andglycosyltransferase(s).

TABLE 4 Modified sugars.

UDP-galactose-derivatives

UDP-galactosaxnine-derivatives (when A = NH, R₄ may be acetyl)

UDP-Glucose-derivatives

UDP-Glucosamine-derivatives (when A = NH, R₄ may be acetyl)

GDP-Mannose-derivatives

GDP-fucose-derivatives X = O, NH, S, CH₂, N-(R_(1–5))₂. Y = X; Z = X; A= X; B = X. Q = H , O, S, NH, N-R. R, R_(1–4) = H, Linker-M, M. M =Ligand of interest Ligand of interest = acyl-PEG, acyl-PPG, alkyl-PEG,acyl-alkyl-PEG, acyl-alkyl-PEG, carbamoyl-PEG, carbamoyl-PPG, PEG, PPG,acyl-aryl-PEG, acyl-aryl-PPG, aryl-PEG, aryl-PPG, Mannose-₆-phosphate,heparin, heparin, SLex, Mannose, FGF, VFGF, protein, chondroitin,keratan, dermatan, albumin, integrins, peptides, etc.

In a further exemplary embodiment, UDP-galactose-PEG is reacted withbovine milk β1,4-galactosyltransferase, thereby transferring themodified galactose to the appropriate terminal N-acetylglucosaminestructure. The terminal GlcNAc residues on the glycopeptide may beproduced during expression, as may occur in such expression systems asmammalian, insect, plant or fungus, but also can be produced by treatingthe glycopeptide with a sialidase and/or glycosidase and/orglycosyltransferase, as required.

In another exemplary embodiment, a GlcNAc transferase, such as GnT-I-IV,is utilized to transfer PEGylated-GlcNc to a mannose residue on aglycopeptide. In a still further exemplary embodiment, the N— and/orO-linked glycan structures are enzymatically removed from a glycopeptideto expose an amino acid or a terminal glycosyl residue that issubsequently conjugated with the modified sugar. For example, anendoglycanase is used to remove the N-linked structures of aglycopeptide to expose a terminal GlcNAc as a GlcNAc-linked-Asn on theglycopeptide. UDP-Gal-PEG and the appropriate galactosyltransferase isused to introduce the PEG- or PPG-galactose functionality onto theexposed GlcNAc.

In an alternative embodiment, the modified sugar is added directly tothe peptide backbone using a glycosyltransferase known to transfer sugarresidues to the peptide backbone. This exemplary embodiment is set forthin Scheme 14. Exemplary glycosyltransferases useful in practicing thepresent invention include, but are not limited to, GalNAc transferases(GalNAc T1-14), GlcNAc transferases, fucosyltransferases,glucosyltransferases, xylosyltransferases, mannosyltransferases and thelike. Use of this approach allows the direct addition of modified sugarsonto peptides that lack any carbohydrates or, alternatively, ontoexisting glycopeptides. In both cases, the addition of the modifiedsugar occurs at specific positions on the peptide backbone as defined bythe substrate specificity of the glycosyltransferase and not in a randommanner as occurs during modification of a protein's peptide backboneusing chemical methods. An array of agents can be introduced intoproteins or glycopeptides that lack the glycosyltransferase substratepeptide sequence by engineering the appropriate amino acid sequence intothe peptide chain.

In each of the exemplary embodiments set forth above, one or moreadditional chemical or enzymatic modification steps can be utilizedfollowing the conjugation of the modified sugar to the peptide. In anexemplary embodiment, an enzyme (e.g., fucosyltransferase) is used toappend a glycosyl unit (e.g., fucose) onto the terminal modified sugarattached to the peptide. In another example, an enzymatic reaction isutilized to “cap” sites to which the modified sugar failed to conjugate.Alternatively, a chemical reaction is utilized to alter the structure ofthe conjugated modified sugar. For example, the conjugated modifiedsugar is reacted with agents that stabilize or destabilize its linkagewith the peptide component to which the modified sugar is attached. Inanother example, a component of the modified sugar is deprotectedfollowing its conjugation to the peptide. One of skill will appreciatethat there is an array of enzymatic and chemical procedures that areuseful in the methods of the invention at a stage after the modifiedsugar is conjugated to the peptide. Further elaboration of the modifiedsugar-peptide conjugate is within the scope of the invention.

Peptide Targeting With Mannose-6-Phosphate

In an exemplary embodiment the peptide is derivatized with at least onemannose-6-phosphate moiety. The mannose-6-phosphate moiety targets thepeptide to a lysosome of a cell, and is useful, for example, to targettherapeutic proteins to lysosomes for therapy of lysosomal storagediseases.

Lysosomal storage diseases are a group of over 40 disorders which arethe result of defects in genes encoding enzymes that break downglycolipid or polysaccharide waste products within the lysosomes ofcells. The enzymatic products, e.g., sugars and lipids, are thenrecycled into new products. Each of these disorders results from aninherited autosomal or X-linked recessive trait which affects the levelsof enzymes in the lysosome. Generally, there is no biological orfunctional activity of the affected enzymes in the cells and tissues ofaffected individuals. Table 5 provides a list of representative storagediseases and the enzymatic defect associated with the diseases. In suchdiseases the deficiency in enzyme function creates a progressivesystemic deposition of lipid or carbohydrate substrate in lysosomes incells in the body, eventually causing loss of organ function and death.The genetic etiology, clinical manifestations, molecular biology andpossibility of the lysosomal storage diseases are detailed in Scriver etal., eds., THE METABOLIC AND MOLECULAR BASIS OF INHERITED DISEASE,7.sup.th Ed., Vol. II, McGraw Hill, (1995).

TABLE 5 Lysosomal storage diseases and associated enzymatic defectsDisease Enzymatic Defect Pompe disease acid α-glucosidase (acid maltase)MPSI* (Hurler disease) α-L-iduronidase MPSII (Hunter disease) iduronatesulfatase MPSIII (Sanfilippo) heparan N-sulfatase MPS IV (Morquio A)galactose-6-sulfatase MPS IV (Morquio B) acid β-galactosidase MPS VII(Sly disease) β-glucoronidase I-cell disease N-acetylglucosamine-1-phosphotransferase Schindler disease α-N-acetylgalactosaminidase(α-galactosidase B) Wolman disease acid lipase Cholesterol ester storagedisease acid lipase Farber disease lysosomal acid ceramidaseNiemann-Pick disease acid sphingomyelinase Gaucher diseaseglucocerebrosidase Krabbe disease galactosylceramidase Fabry diseaseα-galactosidase A GM1 gangliosidosis acid β-galactosidaseGalactosialidosis β-galactosidase and neuraminidase Tay-Sach's diseasehexosaminidase A Magakaryotic leukodystrophy arylsulphatase a Sandhoffdisease hexosaminidase A and B *MPS = mucopolysaccaridosis

De Duve first suggested that replacement of the missing lysosomal enzymewith exogenous biologically active enzyme might be a viable approach totreatment of lysosomal storage diseases (De Duve, Fed. Proc. 23: 1045(1964). Since that time, various studies have suggested that enzymereplacement therapy may be beneficial for treating various lysosomalstorage diseases. The best success has been shown with individuals withtype I Gaucher disease, who have been treated with exogenous enzyme(β-glucocerebrosidase), prepared from placenta (Ceredase™) or, morerecently, recombinantly (Cerezyme™). It has been suggested that enzymereplacement may also be beneficial for treating Fabry's disease, as wellas other lysosomal storage diseases. See, for example, Dawson et al.,Ped. Res. 7(8): 684-690 (1973) (in vitro) and Mapes et al., Science 169:987 (1970) (in vivo). Clinical trials of enzyme replacement therapy havebeen reported for Fabry patients using infusions of normal plasma (Mapeset al., Science 169: 987-989 (1970)), α-galactosidase A purified fromplacenta (Brady et al., N. Eng. J. Med. 279: 1163 (1973)); orα-galactosidase A purified from spleen or plasma (Desnick et al., Proc.Natl. Acad. Sci., USA 76: 5326-5330 (1979)) and have demonstrated thebiochemical effectiveness of direct enzyme replacement for Fabrydisease. These studies indicate the potential for eliminating, orsignificantly reducing, the pathological glycolipid storage by repeatedenzyme replacement. For example, in one study (Desnick et al., supra),intravenous injection of purified enzyme resulted in a transientreduction in the plasma levels of the stored lipid substrate,globotriasylceramide.

Accordingly, there exists a need in the art for methods for providingsufficient quantities of biologically active lysosomal enzymes, such ashuman α-galactosidase A, to deficient cells. Recently, recombinantapproaches have attempted to address these needs, see, e.g., U.S. Pat.Nos. 5,658,567; 5,580,757; Bishop et al., Proc. Natl. Acad. Sci., USA.83: 4859-4863 (1986); Medin et al., Proc. Natl. Acad. Sci., USA. 93:7917-7922 (1996); Novo, F. J., Gene Therapy. 4: 488-492 (1997); Ohshimaet al., Proc. Natl. Acad. Sci., USA. 94: 2540-2544 (1997); and Sugimotoet al., Human Gene Therapy 6: 905-915, (1995). Through themannose-6-phosphate mediated targeting of therapeutic peptides tolysosomes, the present invention provides compositions and methods fordelivering sufficient quantities of biologically active lysosomalpeptides to deficient cells.

Thus, in an exemplary embodiment, the present invention provides apeptide according to Table 7 that is derivatized withmannose-6-phosphate (FIG. 24 and FIG. 25). The peptide may berecombinantly or chemically prepared. Moreover, the peptide can be thefill, natural sequence, or it may be modified by, for example,truncation, extension, or it may include substitutions or deletions.Exemplary proteins that are remodeled using a method of the presentinvention include glucocerebrosidase, β-glucosidase, α-galactosidase A,acid-α-glucosidase (acid maltase). Representative modified peptides thatare in clinical use include, but are not limited to, Ceredase™,Cerezyme™, and Fabryzyme™. A glycosyl group on modified and clinicallyrelevant peptides may also be altered utilizing a method of theinvention. The mannose-6-phosphate is attached to the peptide via aglycosyl linking group. In an exemplary embodiment, the glycosyl linkinggroup is derived from sialic acid. Exemplary siatic acid-derivedglycosyl linking groups are set forth in Table 3, in which one or moreof the “R” moieties is mannose-6-phosphate or a spacer group having oneor more mannose-6-phosphate moieties attached thereto. The modifiedsialic acid moiety is preferably the terminal residue of anoligosaccharide linked to the surface of the peptide (FIG. 26)

In addition to the mannose-6-phosphate, the peptides of the inventionmay be further derivatized with a moiety such as a water-solublepolymer, a therapeutic moiety, or an additional targeting moiety.Methods for attaching these and other groups are set forth herein. In anexemplary embodiment, the group other than mannose-6-phosphate isattached to the peptide via a derivatized sialic acid derivativeaccording to Table 3, in which one or more of the “R” moieties is agroup other than mannose-6-phosphate.

In an exemplary embodiment, a sialic acid moiety modified with aCbz-protected glycine-based linker arm is prepared. The correspondingnucleotide sugar is prepared and the Cbz group is removed by catalytichydrogenation. The resulting nucleotide sugar has an available, reactiveamine that is contacted with an activated mannose-6-phosphatederivative, providing a mannose-6-phosphate derivatized nucleotide sugarthat is useful in practicing the methods of the invention.

As shown in the scheme below (scheme 15), an exemplary activatedmannose-6-phosphate derivative is formed by converting a2-bromo-benzyl-protected phosphotriester into the correspondingtriflate, in situ, and reacting the triflate with a linker having areactive oxygen-containing moiety, forming an ether linkage between thesugar and the linker. The benzyl protecting groups are removed bycatalytic hydrogenation, and the methyl ester of the linker ishydrolyzed, providing the corresponding carboxylic acid. The carboxylicacid is activated by any method known in the art. An exemplaryactivation procedure relies upon the conversion of the carboxylic acidto the N-hydroxysuccinimide ester.

In another exemplary embodiment, as shown in the scheme below (scheme16), a N-acetylated sialic acid is converted to an amine by manipulationof the pyruvyl moiety. Thus, the primary hydroxyl is converted to asulfonate ester and reacted with sodium azide. The azide iscatalytically reduced to the corresponding amine. The sugar issubsequently converted to its nucleotide analogue and coupled, throughthe amine group, to the linker arm-derivatized mannose-6-phosphateprepared as discussed above.

Peptides useful to treat lysosomal storage disease can be derivatizedwith other targeting moieties including, but not limited to, transferrin(to deliver the peptide across the blood-brain barrier, and toendosomes), carnitine (to deliver the peptide to muscle cells), andphosphonates, e.g, bisphosphonate (to target the peptide to bone andother calciferous tissues). The targeting moiety and therapeutic peptideare conjugated by any method discussed herein or otherwise known in theart.

In an exemplary embodiment, the targeting agent and the therapeuticpeptide are coupled via a linker moiety. In this embodiment, at leastone of the therapeutic peptide or the targeting agent is coupled to thelinker moiety via an intact glycosyl linking group according to a methodof the invention. In an exemplary embodiment, the linker moiety includesa poly(ether) such as poly(ethylene glycol). In another exemplaryembodiment, the linker moiety includes at least one bond that isdegraded in vivo, releasing the therapeutic peptide from the targetingagent, following delivery of the conjugate to the targeted tissue orregion of the body.

In yet another exemplary embodiment, the in vivo distribution of thetherapeutic moiety is altered via altering a glycoform on thetherapeutic moiety without conjugating the therapeutic peptide to atargeting moiety. For example, the therapeutic peptide can be shuntedaway from uptake by the reticuloendothelial system by capping a terminalgalactose moiety of a glycosyl group with sialic acid (or a derivativethereof) (FIGS. 24 and 27). Sialylation to cover terminal Gal avoidsuptake of the peptide by hepatic asialoglycoprotein (ASGP) receptors,and may extend the half life of the peptide as compared with peptideshaving only complex glycan chains, in the absence of sialylation.

II. Peptide/Glycopeptides of the Invention

In one embodiment, the present invention provides a compositioncomprising multiple copies of a single peptide having an elementaltrimannosyl core as the primary glycan structure attached thereto. Inpreferred embodiments, the peptide may be a therapeutic molecule. Thenatural form of the peptide may comprise complex N-linked glycans or maybe a high mannose glycan. The peptide may be a mammalian peptide, and ispreferably a human peptide. In some embodiments the peptide is selectedfrom the group consisting of an immunoglobulin, erythropoietin,tissue-type activator peptide, and others (See FIG. 28).

Exemplary peptides whose glycans can be remodeled using the methods ofthe invention are set forth in FIG. 28.

TABLE 6 Preferred peptides for glycan remodeling Hormones and GrowthFactors G-CSF GM-CSF TPO EPO EPO variants FSH HGH insulin alpha-TNFLeptin human chorionic gonadotropin Enzymes and Inhibitors TPA TPAvariants Urokinase Factors VII, VIII, IX, X DNase GlucocerebrosidaseHirudin α1 antitrypsin (α1 protease inhibitor) Antithrombin III Acidα-glucosidase (acid maltase) α galactosidase A α-L-iduronidase UrokinaseCytokines and Chimeric Cytokines Interleukin-1 (IL-1), 1B, 2, 3, 4Interferon-alpha (IFN-alpha) IFN-alpha-2b IFN-beta IFN-gamma IFN-omegaChimeric diphtheria toxin-IL-2 Receptors and Chimeric Receptors CD4Tumor Necrosis Factor receptor (TNF-R) TNF-R: IgG Fc fusion Alpha-CD20PSGL-1 Complement GlyCAM or its chimera N-CAM or its chimera MonoclonalAntibodies (Immunoglobulins) MAb-anti-RSV MAb-anti-IL-2 receptorMAb-anti-CEA MAb-anti-glycoprotein IIb/IIIa MAb-anti-EGF MAb-anti-Her2MAb-CD20 MAb-alpha-CD3 MAb-TNFα MAb-CD4 MAb-PSGL-1 Mab-anti F protein ofRespiratory Syncytial Virus Anti-thrombin-III Cells Red blood cellsWhite blood cells (e.g., T cells, B cells, dendritic cells, macrophages,NK cells, neutrophils, monocytes and the like) Stem cells OthersHepatits B surface antigen (HbsAg)

TABLE 7 Most preferred peptides for glycan remodelingAlpha-galactosidase A Interleukin-2 (IL-2) Alpha-L-iduronidase FactorVIII Anti-thrombin-III hrDNase Granulocyte colony Insulin stimulatingfactor (G-CSF) Hepatitis B surface protein (HbsAg) Interferon α HumanGrowth Hormone (HGH) Interferon β Human chorionic gonadotropinInterferon omega Urokinase Factor VII clotting factor TNF receptor-IgGFc fusion (Enbrel ™) Factor IX clotting factor MAb-Her-2 (Herceptin ™)Follicle Stimulating MAb-F protein Hormone (FSH) of RespiratoryErythropoietin (EPO) Syncytial Virus (Synagis ™) Granulocyte-macrophagecolony MAb-CD20 (Rituxan ™) stimulating factor (GM-CSF) MAb-TNFα(Remicade ™) Interferon γ MAb-Glycoprotein IIb/IIIa (Reopro ™) α₁protease inhibitor (α₁ antitrypsin) Tissue-type plasminogen activator(TPA) Glucocerebrosidase (Cerezyme ™)

A more detailed list of peptides useful in the invention and theirsource is provided in FIG. 28.

Other exemplary peptides that are modified by the methods of theinvention include members of the immunoglobulin family (e.g.,antibodies, MHC molecules, T cell receptors, and the like),intercellular receptors (e.g., integrins, receptors for hormones orgrowth factors and the like) lectins, and cytokines (e.g.,interleukins). Additional examples include tissue-type plasminogenactivator (TPA), renin, clotting factors such as Factor VIII and FactorIX, bombesin, thrombin, hematopoietic growth factor, colony stimulatingfactors, viral antigens, complement peptides, α1-antitrypsin,erythropoietin, P-selectin glycopeptide ligand-l (PSGL-1),granulocyte-macrophage colony stimulating factor, anti-thrombin III,interleukins, interferons, peptides A and C, fibrinogen, herceptin™,leptin, glycosidases, among many others. This list of peptides isexemplary and should not be considered to be exclusive. Rather, as isapparent from the disclosure provided herein, the methods of theinvention are applicable to any peptide in which a desired glycanstructure can be fashioned.

The methods of the invention are also useful for modifying chimericpeptides, including, but not limited to, chimeric peptides that includea moiety derived from an immunoglobulin, such as IgG.

Peptides modified by the methods of the invention can be synthetic orwild-type peptides or they can be mutated peptides, produced by methodsknown in the art, such as site-directed mutagenesis. Glycosylation ofpeptides is typically either N-linked or O-linked. An exemplaryN-linkage is the attachment of the modified sugar to the side chain ofan asparagine residue. The tripeptide sequences asparagine-X-serine andasparagine-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of a carbohydratemoiety to the asparagine side chain. Thus, the presence of either ofthese tripeptide sequences in a peptide creates a potentialglycosylation site. As described elsewhere herein, O-linkedglycosylation refers to the attachment of one sugar (e.g.,N-acetylgalactosamine, galactose, mannose, GlcNAc, glucose, fucose orxylose) to a hydroxy side chain of a hydroxyamino acid, preferablyserine or threonine, although 5-hydroxyproline or 5-hydroxylysine mayalso be used.

Several exemplary embodiments of the invention are discussed below.While several of these embodiments use peptides having names havingtrademarks, and other specific peptides as the exemplary peptide, theseexamples are not confined to any specific peptide. The followingexemplary embodiments are contemplated to include all peptideequivalents and variants of any peptide. Such variants include, but arenot limited to, adding and deleting N-linked and O-linked glycosylationsites, and fusion proteins with added glycosylation sites. One of skillin the art will appreciate that the following embodiments and the basicmethods disclosed therein can be applied to many peptides with equalsuccess.

In one exemplary embodiment, the present invention provides methods formodifying Granulocyte Colony Stimulating Factor (G-CSF). FIGS. 29A to29G set forth some examples of how this is accomplished using themethodology disclosed herein. In FIG. 29B, a G-CSF peptide that isexpressed in a mammalian cell system is trimmed back using a sialidase.The residues thus exposed are modified by the addition of a sialicacid-poly(ethylene glycol) moiety (PEG moiety), using an appropriatedonor therefor and ST3Gal1. FIG. 29C sets forth an exemplary scheme formodifing a G-CSF peptide that is expressed in an insect cell. Thepeptide is modified by adding a galactose moiety using an appropriatedonor thereof and a galactosyltransferase. The galactose residues arefunctionalized with PEG via a sialic acid-PEG derivative, through theaction of ST3Gal1. In FIG. 29D, bacterially expressed G-CSF is contactedwith an N-acetylgalactosamine donor and N-acetylgalactosaminetransferase. The peptide is functionalized with PEG, using a PEGylatedsialic acid donor and a sialyltransferase. In FIG. 29E, mammalian cellexpressed G-CSF is contacted with a sialic acid donor that is modifiedwith levulinic acid, adding a reactive ketone to the sialic acid donor.After addition to a glycosyl residue on the glycan on the peptide, theketone is derivatized with a moiety such as a hydrazine- or amine-PEG.In FIG. 29F, bacterially expressed G-CSF is remodeled by contacting thepeptide with an endo-GalNAc enzyme under conditions where it functionsin a synthetic, rather than a hydrolytic manner, thereby adding aPEG-Gal-GalNAc molecule from an activated derivative thereof. FIG. 29Gprovides another route for remodeling bacterially expressed G-CSF. Thepolypeptide is derivatized with a PEGylated N-acetylgalactosamineresidue by contacting the polypeptide with an N-acetylgalactosaminetransferase and an appropriate donor of PEGylated N-acetylgalactosanine.

In another exemplary embodiment, the invention provides methods formodifying Interferon α-14C (IFNα14C), as shown in FIGS. 30A to 30N. Thevarious forms of IFNα are disclosed elsewhere herein. In FIG. 30B,IFNα14C expressed in mammalian cells is first treated with sialidase totrim back the sialic acid units thereon, and then the molecule isPEGylated using ST3Gal3 and a PEGylated sialic acid donor. In FIG. 30C,N-acetylglucosamine is first added to IFNα14C which has been expressedin insect or fungal cells, where the reaction is conducted via theaction of GnT-I and/or II using an N-acetylglucosamine donor. Thepolypeptide is then PEGylated using a galactosyltransferase and a donorof PEG-galactose. In FIG. 30D, IFNα14C expressed in yeast is firsttreated with Endo-H to trim back the glycosyl units thereon. Themolecules is galactosylated using a galactosyltransferase and agalactose donor, and it is then PEGylated using ST3Gal3 and a donor ofPEG-sialic acid. In FIG. 30F, IFNα14C produced by mammalian cells ismodified to inched a PEG moiety using ST3Gal3 and, a donor of PEG-sialicacid. In FIG. 30G, IFNα14C expressed in insect of fungal cells first hasN-acetylglucosamine added using one or more of GnT-I, II, IV, and V, andan N-acetylglucosamine donor. The protein is subsequently galactosylatedusing an appropriate donor and a galactosyltransferase. Then, IFNα14C isPEGylated using ST3Gal3 and a donor of PEG-sialic acid. In FIG. 30H,yeast produced IFNα14C is first treated with mannosidases to trim backthe mannosyl groups. N-acetylglucosamine is then added using a donor ofN-acetylglucosamine and one or more of GnT-I, II, IV, and V. IFNα14C isfurther galactosylated using an appropriate donor and agalactosyltransferase. Then, the polypeptide is PEGylated using ST3Gal3and a donor of PEG-sialic acid. In FIG. 30I, NSO cell expressed IFNα14Cis modified by capping appropriate terminal residues with a sialic aciddonor that is modified with levulinic acid, thereby adding a reactiveketone to the sialic acid donor. After addition to a glycosyl residue ofthe peptide, the ketone is derivatized with a moiety such as ahydrazine- or amine-PEG. In FIG. 30J, IFNα14C expressed by mammaliancells is PEGylated using a donor of PEG-sialic acid andα2,8-sialyltransferase. In FIG. 30K, IFNα14C produced by mammalian cellsis first treated with sialidase to trim back the terminal sialic acidresidues, and then the molecule is PEGylated using trans-sialidase andPEGylated sialic acid-lactose complex. In FIG. 30L, IFNα14C expressed ina mammalian system is sialylated using a donor of sialic acid andα2,8-sialyltransferase. In FIG. 30M, IFNα14C expressed in insect orfungal cells first has N-acetylglucosamine added using an appropriatedonor and GnT-I and/or II. The molecule is then contacted with agalactosyltransferase and a galactose donor that is derivatized with areactive sialic acid via a linker, so that the polypeptide is attachedto the reactive sialic acid via the linker and the galactose residue.The polypeptide is then contacted with ST3Gal3 and transferrin, and thusbecomes connected with transferrin via the sialic acid residue. In FIG.30N, IFNα14C expressed in either insect or fungal cells is first treatedwith endoglycanase to trim back the glycosyl groups, and is thencontacted with a galactosyltransferase and a galactose donor that isderivatized with a reactive sialic acid via a linker, so that thepolypeptide is attached to the reactive sialic acid via the linker andthe galactose residue. The molecule is then contacted with ST3Gal3 andtransferrin, and thus becomes connected with transferrin via the sialicacid residue.

In another exemplary embodiment, the invention provides methods formodifing Interferon α-2a or 2b (IFNα), as shown in FIGS. 30O to 30EE. InFIG. 30P, IFNα produced in mammalian cells is first treated withsialidase to trim back the glycosyl units, and is then PEGylated usingST3Gal3 and a PEGylated sialic acid donor. In FIG. 30Q, IFNα expressedin insect cells is first galactosylated using an appropriate donor and agalactosyltransferase, and is then PEGylated using ST3Gal1 and aPEGylated sialic acid donor. FIG. 30R offers another method forremodeling IFNα expressed in bacteria: PEGylated N-acetylgalactosamineis added to the protein using an appropriate donor andN-acetylgalactosamine transferase. In FIG. 30S, IFNα expressed inmammalian cells is modified by capping appropriate terminal residueswith a sialic acid donor that is modified with levulinic acid, adding areactive ketone to the sialic acid donor. After addition to a glycosylresidue of the peptide, the ketone is derivatized with a moiety such asa hydrazine- or amine-PEG. In FIG. 30T, IFNα expressed in bacteria isPEGylated using a modified enzyme Endo-N-acetylgalactosamidase, whichfunctions in a synthetic instead of a hydrolytic manner, and using aN-acetylgalactosamine donor derivatized with a PEG moiety. In FIG. 30U,N-acetylgalactosamine is first added IFNα using an appropriate donor andN-acetylgalactosamine transferase, and then is PEGylated using asialyltransferase and a PEGylated sialic acid donor. In FIG. 30V, IFNαexpressed in a mammalian system is first treated with sialidase to trimback the sialic acid residues, and is then PEGylated using a suitabledonor and ST3Gal1 and/or ST3Gal3. In FIG. 30W, IFNα expressed inmammalian cells is first treated with sialidase to trim back the sialicacid residues. The polypeptide is then contacted with ST3Gal1 and tworeactive sialic acid residues that are connect via a linker, so that thepolypeptide is attached to one reactive sialic acid via the linker andthe second sialic acid residue. The polypeptide is subsequentlycontacted with ST3Gal3 and transferrin, and thus becomes connected withtransferrin via the sialic acid residue. In FIG. 30Y, IFNα expressed inmammalian cells is first treated with sialidase to trim back the sialicacid residues, and is then PEGylated using ST3Gal1 and a donor ofPEG-sialic acid. In FIG. 30Z, IFNα produced by insect cells is PEGylatedusing a galactosyltransferase and a donor of PEGylated galactose. InFIG. 30AA, bacterially expressed IFNα first has N-acetylgalactosamineadded using a suitable donor and N-acetylgalactosamine transferase. Theprotein is then PEGylated using a sialyltransferase and a donor ofPEG-sialic acid. In FIG. 30CC, IFNα expressed in bacteria is modified inanother procedure: PEGylated N-acetylgalactosamine is added to theprotein by N-acetylgalactosamine transferase using a donor of PEGylatedN-acetylgalactosamine. In FIG. 30DD, IFNα expressed in bacteria isremodeled in yet another scheme. The polypeptide is first contacted withN-acetylgalactosamine transferase and a donor of N-acetylgalactosaminethat is derivatized with a reactive sialic acid via a linker, so thatIFNα is attached to the reactive sialic acid via the linker and theN-acetylgalactosamine. IFNα is then contacted with ST3Gal3 andasialo-transferrin so that it becomes connected with transferrin via thesialic acid residue. Then, IFNα is capped with sialic acid residuesusing ST3Gal3 and a sialic acid donor. An additional method formodifying bacterially expressed IFNα is disclosed in FIG. 30EE, whereIFNα is first exposed to NHS-CO-linker-SA-CMP and is then connected to areactive sialic acid via the linker. It is subsequently conjugated withtransferrin using ST3Gal3 and transferrin.

The methods for remodeling INN omega are essentially identical to thosepresented here for IFN alpha except that the attachment of the glycan tothe IFN omega peptide occurs at amino acid residue 101 in SEQ ID NO:75.The nucleotide and amino acid sequences for IFN omega are presentedherein as SEQ ID NOS:74 and 75. Methods of making and using IFN omegaare found in U.S. Pat. Nos. 4,917,887 and 5,317,089, and in EP PatentNo. 0170204-A.

In another exemplary embodiment, the invention provides methods formodifying Interferon β (IFN-β), as shown in FIGS. 31A to 31S. In FIG.31B, IFN-β expressed in a mammalian system is first treated withsialidase to trim back the terminal sialic acid residues. The protein isthen PEGylated using ST3Gal3 and a donor of PEGylated sialic acid. FIG.31C is a scheme for modifying IFN-β produced by insect cells. First,N-acetylglucosamine is added to IFN-β using an appropriate donor andGnT-I and/or -II. The protein is then galactosylated using a galactosedonor and a galactosyltransferase. Finally, IFN-β is PEGylated usingST3Gal3 and a donor of PEG-sialic acid. In FIG. 31D, IFN-β expressed inyeast is first treated with Endo-H to trim back its glycosyl chains, andis then galactosylated using a galactose donor and agalactosyltransferase, and is then PEGylated using ST3Gal3 and a donorof PEGylated sialic acid. In FIG. 31E, IFN-β produced by mammalian cellsis modified by PEGylation using ST3Gal3 and a donor of sialic acidalready derivatized with a PEG moiety. In FIG. 31F, IFN-β expressed ininsect cells first has N-acetylglucosamine added by one or more ofGnT-I, II, IV, and V using a N-acetylglucosamine donor, and then isgalactosylated using a galactose donor and a galactosyltransferase, andis then PEGylated using ST3Gal3 and a donor of PEG-sialic acid. In FIG.31G, IFN-β expressed in yeast is first treated with mannosidases to trimback the mannosyl units, then has N-acetylglucosamine added using aN-acetylglucosamine donor and one or more of GnT-I, II, IV, and V. Theprotein is further galactosylated using a galactose donor and agalactosyltransferase, and then PEGylated using ST3Gal3 and a PEG-sialicacid donor. In FIG. 31H, mammalian cell expressed IFN-β is modified bycapping appropriate terminal residues with a sialic acid donor that ismodified with levulinic acid, adding a reactive ketone to the sialicacid donor. After addition to a glycosyl residue of the peptide, theketone is derivatized with a moiety such as a hydrazine- or amine-PEG.In FIG. 31I, IFN-β expressed in a mammalian system is PEGylated using adonor of PEG-sialic acid and α2,8-sialyltransferase. In FIG. 31J, IFN-βexpressed by mammalian cells is first treated with sialidase to trimback its terminal sialic acid residues, and then PEGylated usingtrans-sialidase and a donor of PEGylated sialic acid. In FIG. 31K, IFN-βexpressed in mammalian cells is first treated with sialidase to trimback terminal sialic acid residues, then PEGylated using ST3Gal3 and adonor of PEG-sialic acid, and then sialylated using ST3Gal3 and a sialicacid donor. In FIG. 31L, IFN-β expressed in mammalian cells is firsttreated with sialidase and galactosidase to trim back the glycosylchains, then galactosylated using a galactose donor and anα-galactosyltransferase, and then PEGylated using ST3Gal3 or asialyltransferase and a donor of PEG-sialic acid. In FIG. 31M, IFN-βexpressed in mammalian cells is first treated with sialidase to trimback the glycosyl units. It is then PEGylated using ST3Gal3 and a donorof PEG-sialic acid, and is then sialylated using ST3Gal3 and a sialicacid donor. In FIG. 31N, IFN-β expressed in mammalian cells is modifiedby capping appropriate terminal residues with a sialic acid donor thatis modified with levulinic acid, adding a reactive ketone to the sialicacid donor. After addition to a glycosyl residue of the peptide, theketone is derivatized with a moiety such as a hydrazine- or amine-PEG.In FIG. 31O, IFN-β expressed in mammalian cells is sialylated using asialic acid donor and α2,8-sialyltransferase. In FIG. 31Q, IFN-βproduced by insect cells first has N-acetylglucosamine added using adonor of N-acetylglucosamine and one or more of GnT-I, II, IV, and V,and is further PEGylated using a donor of PEG-galactose and agalactosyltransferase. In FIG. 31R, IFN-β expressed in yeast is firsttreated with endoglycanase to trim back the glycosyl groups, thengalactosylated using a galactose donor and a galactosyltransferase, andthen PEGylated using ST3Gal3 and a donor of PEG-sialic acid. In FIG.31S, IFN-β expressed in a mammalian system is first contacted withST3Gal3 and two reactive sialic acid residues connected via a linker, sothat the polypeptide is attached to one reactive sialic acid via thelinker and the second sialic acid residue. The polypeptide is thencontacted with ST3Gal3 and desialylated transferrin, and thus becomesconnected with transferrin via the sialic acid residue. Then, IFN-β isfurther sialylated using a sialic acid donor and ST3Gal3.

In another exemplary embodiment, the invention provides methods formodifying Factor VII or VIIa, as shown in FIGS. 32A to 32D. In FIG. 32B,Factor VII or VIIa produced by a mammalian system is first treated withsialidase to trim back the terminal sialic acid residues, and thenPEGylated using ST3Gal3 and a donor of PEGylated sialic acid. FIG. 32C,Factor VII or VIIa expressed by mammalian cells is first treated withsialidase to trim back the terminal sialic acid residues, and thenPEGylated using ST3Gal3 and a donor of PEGylated sialic acid. Further,the polypeptide is sialylated with ST3Gal3 and a sialic acid donor. FIG.32D offers another modification scheme for Factor VII or VIIa producedby mammalian cells: the polypeptide is first treated with sialidase andgalactosidase to trim back its sialic acid and galactose residues, thengalactosylated using a galactosyltransferase and a galactose donor, andthen PEGylated using ST3Gal3 and a donor of PEGylated sialic acid.

In another exemplary embodiment, the invention provides methods formodifying Factor IX, some examples of which are included in FIGS. 33A to33G. In FIG. 33B, Factor IX produced by mammalian cells is first treatedwith sialidase to trim back the terminal sialic acid residues, and isthen PEGylated with ST3Gal3 using a PEG-sialic acid donor. In FIG. 33C,Factor IX expressed by mammalian cells is first treated with sialidaseto trim back the terminal sialic acid residues, it is then PEGylatedusing ST3Gal3 and a PEG-sialic acid donor, and further sialylated usingST3Gal1 and a sialic acid donor. Another scheme for remodeling mammaliancell produced Factor IX can be found in FIG. 33D. The polypeptide isfirst treated with sialidase to trim back the terminal sialic acidresidues, then galactosylated using a galactose donor and agalactosyltransferase, further sialylated using a sialic acid donor andST3Gal3, and then PEGylated using a donor of PEGylated sialic acid andST3Gall. In FIG. 33E, Factor IX that is expressed in a mammalian systemis PEGylated through the process of sialylation catalyzed by ST3Gal3using a donor of PEG-sialic acid. In FIG. 33F, Factor IX expressed inmammalian cells is modified by capping appropriate terminal residueswith a sialic acid donor that is modified with levulinic acid, adding areactive ketone to the sialic acid donor. After addition to a glycosylresidue of the peptide, the ketone is derivatized with a moiety such asa hydrazine- or amine-PEG. FIG. 33G provides an additional method ofmodifying Factor IX. The polypeptide, produced by mammalian cells, isPEGylated using a donor of PEG-sialic acid and α2,8-sialyltransferase.

In another exemplary embodiment, the invention provides methods formodification of Follicle Stimulating Hormone (FSH). FIGS. 34A to 34Jpresent some examples. In FIG. 34B, FSH is expressed in a mammaliansystem and modified by treatment of sialidase to trim back terminalsialic acid residues, followed by PEGylation using ST3Gal3 and a donorof PEG-sialic acid. In FIG. 34C, FSH expressed in mammalian cells isfirst treated with sialidase to trim back terminal sialic acid residues,then PEGylated using ST3Gal3 and a donor of PEG-sialic acid, and thensialylated using ST3Gal3 and a sialic acid donor. FIG. 34D provides ascheme for modifying FSH expressed in a mammalian system. Thepolypeptide is treated with sialidase and galactosidase to trim back itssialic acid and galactose residues, then galactosylated using agalactose donor and a galactosyltransferase, and then PEGylated usingST3Gal3 and a donor of PEG-sialic acid. In FIG. 34E, FSH expressed inmammalian cells is modified in the following procedure: FSH is firsttreated with sialidase to trim back the sialic acid residues, thenPEGylated using ST3Gal3 and a donor of PEG-sialic acid, and is thensialylated using ST3Gal3 and a sialic acid donor. FIG. 34F offersanother example of modifying FSH produced by mammalian cells: Thepolypeptide is modified by capping appropriate terminal residues with asialic acid donor that is modified with levulinic acid, adding areactive ketone to the sialic acid donor. After addition to a glycosylresidue of the peptide, the ketone is derivatized with a moiety such asa hydrazine- or amine-PEG. In FIG. 34G, FSH expressed in a mammaliansystem is modified in another procedure: the polypeptide is remodeledwith addition of sialic acid using a sialic acid donor and anα2,8-sialyltransferase. In FIG. 34H, FSH is expressed in insect cellsand modified in the following procedure: N-acetylglucosamine is firstadded to FSH using an appropriate N-acetylglucosamine donor and one ormore of GnT-I, II, IV, and V; FSH is then PEGylated using a donor ofPEG-galactose and a galactosyltransferase. FIG. 34I depicts a scheme ofmodifying FSH produced by yeast. According to this scheme, FSH is firsttreated with endoglycanase to trim back the glycosyl groups,galactosylated using a galactose donor and a galactosyltransferase, andis then PEGylated with ST3Gal3 and a donor of PEG-sialic acid. In FIG.34J, FSH expressed by mammalian cells is first contacted with ST3Gal3and two reactive sialic acid residues via a linker, so that thepolypeptide is attached to a reactive sialic acid via the linker and asecond sialic acid residue. The polypeptide is then contacted withST3Gal1 and desialylated chorionic gonadotrophin (CG) produced in CHO,and thus becomes connected with CG via the second sialic acid residue.Then, FSH is sialylated using a sialic acid donor and ST3Gal3 and/orST3Gal1.

In another exemplary embodiment, the invention provides methods formodifying erythropoietin (EPO), FIGS. 35A to 35AA set forth someexamples which are relevant to the remodeling of both wild-type andmutant EPO peptides. In FIG. 35B, EPO expressed in various mammaliansystems is remodeled by contacting the expressed protein with asialidase to remove terminal sialic acid residues. The resulting peptideis contacted with a sialyltransferase and a CMP-sialic acid that isderivatized with a PEG moiety. In FIG. 35C, EPO that is expressed ininsect cells is remodeled with N-acetylglucosamine, using GnT-I and/orGnT-II. Galactose is then added to the peptide, usinggalactosyltransferase. PEG group is added to the remodeled peptide bycontacting it with a sialyltransferase and a CMP-sialic acid that isderivatized with a PEG moiety. In FIG. 35D, EPO that is expressed in amammalian cell system is remodeled by removing terminal sialic acidmoieties via the action of a sialidase. The terminal galactose residuesof the N-linked glycosyl units are “capped” with sialic acid, usingST3Gal3 and a sialic acid donor. The terminal galactose residues on theO-linked glycan are functionalized with a sialic acid bearing a PEGmoiety, using an appropriate sialic acid donor and ST3Gall. In FIG. 35E,EPO that is expressed in a mammalian cell system is remodeled byfunctionalizing the N-linked glycosyl residues with a PEG-derivatizedsialic acid moiety. The peptide is contacted with ST3Gal3 and anappropriately modified sialic acid donor. In FIG. 35F, EPO that isexpressed in an insect cell system, yeast or fungi, is remodeled byadding at least one N-acetylglucosamine residues by contacting thepeptide with a N-acetylglucosamine donor and one or more of GnT-I,GnT-II, and GnT-V. The peptide is then PEGylated by contacting it with aPEGylated galactose donor and a galactosyltransferase. In FIG. 35G, EPOthat is expressed in an insect cell system, yeast or fungi, is remodeledby the addition of at least one N-acetylglucosamine residues, using anappropriate N-acetylglucosamine donor and one or more of GnT-I, GnT-II,and GnT-V. A galactosidase that is altered to operate in a synthetic,rather than a hydrolytic manner is used to add an activated PEGylatedgalactose donor to the N-acetylglucosamine residues. In FIG. 35H, EPOthat is expressed in an insect cell system, yeast or fungi, is remodeledby the addition of at least one terminal N-acetylglucosamine-PEGresidue. The peptide is contacted with GnT-I and an appropriateN-acetlyglucosamine donor that is derivatized with a PEG moiety. In FIG.35I, EPO that is expressed in an insect cell system, yeast or fungi, isremodeled by adding one or more terminal galactose-PEG residues. Thepeptide is contacted with GnT-I and an appropriate N-acetylglucosaminedonor that is derivatized with a PEG-moiety. The peptide is thencontacted with galactosyltransferase and an appropriate galactose donorthat is modified with a PEG moiety. In FIG. 35J, EPO expressed in aninsect cell system, yeast or fungi, is remodeled by the addition of onemore terminal sialic acid-PEG residues. The peptide is contacted with anappropriate N-acetylglucosamine donor and GnT-I. The peptide is furthercontacted with galactosyltransferase and an appropriate galactose donor.The peptide is then contacted with ST3Gal3 and an appropriate sialicacid donor that is derivatized with a PEG moiety. In FIG. 35K, EPOexpressed in an insect cell system, yeast or fungi, is remodeled by theaddition of terminal sialic acid-PEG residues. The peptide is contactedwith an appropriate N-acetylglucosamine donor and one or more of GnT-I,GnT-II, and GnT-V. The peptide is then contacted withgalactosyltransferase and an appropriate galactose donor. The peptide isfurther contacted with ST3Gal3 and an appropriate sialic acid donor thatis derivatized with a PEG moiety. In FIG. 35L, EPO expressed in aninsect cell system, yeast or fungi, is remodeled by the addition of oneor more terminal α2,6-sialic acid-PEG residues. The peptide is contactedwith an appropriate N-acetylglucosamine donor and one or more of GnT-I,GnT-II, and GnT-V. The peptide is further contacted withgalactosyltransferase and an appropriate galactose donor. The peptide isthen contacted with α2,6-sialyltransferase and an appropriately modifiedsialic acid donor. In FIG. 35M, EPO expressed in a mammalian cell systemis remodeled by addition of one or more terminal sialic acid-PEGresidues. The peptide is contacted with a sialidase to remove terminalsialic acid residues. The peptide is further contacted with asialyltransferase and an appropriate sialic acid donor. The peptide isfurther contacted with a sialyltransferase and an appropriate sialicacid donor that is derivatized with a PEG moiety. In FIG. 35N, EPOexpressed in a mammalian cell system is remodeled by the addition of oneor more terminal sialic acid-PEG residues. The peptide is contacted witha sialyltransferase and an appropriate sialic acid donor that isderivatized with a PEG moiety. In FIG. 35O, EPO expressed in a mammaliancell system is remodeled by the addition of one or more terminalα2,8-sialic acid-PEG residues to primarily O-linked glycans. The peptideis contacted with α2,8-sialyltransferase and an appropriate sialic aciddonor that is derivatized with a PEG moiety. In FIG. 35P, EPO expressedin a mammalian cell is remodeled by the addition of one or more terminalα2,8-sialic acid-PEG residues to O-linked and N-linked glycans. Thepeptide is contacted with α2,8-sialyltransferase and an appropriatesialic acid donor that is derivatized with a PEG moiety. In FIG. 35Q,EPO expressed in yeast or fungi is remodeled by the addition of one ormore terminal sialic acid-PEG residues. The peptide is contacted withmannosidases to remove terminal mannose residues. Next, the peptide iscontacted with GnT-I and an appropriate N-acetylglucosamine donor. Thepeptide is further contacted with galactosyltransferase and anappropriate galactose donor. The peptide is then contacted with asialyltransferase and an appropriate sialic acid donor that isderivatized with a PEG moiety. In FIG. 35R, EPO expressed in yeast orfungi is remodeled by the addition of at least one terminalN-acetylglucosamine-PEG residues. The peptide is contacted withmannosidases to remove terminal mannose residue. The peptide is thencontacted with GnT-I and an appropriate N-acetylglucosamine donor thatis derivatized with a PEG moiety. In FIG. 35S, EPO expressed in yeast orfungi is remodeled by the addition of one or more terminal sialicacid-PEG residues. The peptide is contacted with mannosidase-I to removeα2 mannose residues. The peptide is further contacted with GnT-I and anappropriate N-acetylglucosamine donor. The peptide is then contactedwith galactosyltransferase and an appropriate galacose donor. Thepeptide is then contacted with a sialyltransferase and an appropriatesialic acid donor that is derivatized with a PEG moiety. In FIG. 35U,EPO expressed in yeast or fungi is remodeled by addition of one or moregalactose-PEG residues. The peptide is contacted with endo-H to trimback glycosyl groups. The peptide is then contacted withgalactosyltransferase and an appropriate galactose donor that isderivatized with a PEG moiety. In FIG. 35V, EPO expressed in yeast orfungi is remodeled by the addition of one or more terminal sialicacid-PEG residues. The peptide is contacted with endo-H to trim backglycosyl groups. The peptide is further contacted withgalactosyltransferase and an appropriate galactose donor. The peptide isthen contacted with a sialyltransferase and an appropriate sialic aciddonor that is derivatized with a PEG moiety. In FIG. 35W, EPO expressedin an insect cell system is remodeled by the addition of terminalgalactose-PEG residues. The peptide is contacted with mannosidases toremove terminal mannose residues. The peptide is then contacted withgalactosyltransferase and an appropriate galactose donor that isderivatized with a PEG moeity. In FIG. 35Y, a mutant EPO called “novelerythropoiesis-stimulating protein” or NESP, expressed in NSO murinemyeloma cells is remodeled by capping appropriate terminal residues witha sialic acid donor that is modified with levulinic acid, adding areactive ketone to the sialic acid donor. After addition to a glycosylresidue of the peptide, the ketone is derivatized with a moiety such asa hydrazine- or amine-PEG. In FIG. 35Z, mutant EPO, i.e. NESP, expressedin a mammalian cell system is remodeled by addition of one or moreterminal sialic acid-PEG residues. PEG is added to the glycosyl residueon the glycan using a PEG-modified sialic acid and anα2,8-sialyltransferase. In FIG. 35AA, NESP expressed in a mammalian cellsystem is remodeled by the addition of terminal sialic acid residues.The sialic acid is added to the glycosyl residue using a sialic aciddonor and an α2,8-sialyltransferase.

In another exemplary embodiment, the invention provides methods formodifying granulocyte-macrophage colony-stimulating factor (GM-CSF), asshown in FIGS. 36A to 36K. In FIG. 36B, GM-CSF expressed in mammaliancells is first treated with sialidase to trim back the sialic acidresidues, and then PEGylated using ST3Gal3 and a donor of PEG-sialicacid. In FIG. 36C, GM-CSF expressed in mammalian cells is first treatedwith sialidase to trim back the sialic acid residues, then PEGylatedusing ST3Gal3 and a donor of PEG-sialic acid, and then is furthersialylated using a sialic acid donor and ST3Gal1 and/or ST3Gal3. In FIG.36D, GM-CSF expressed in NSO cells is first treated with sialidase andα-galactosidase to trim back the glycosyl groups, then sialylated usinga sialic acid donor and ST3Gal3, and is then PEGylated using ST3Gal1 anda donor of PEG-sialic acid. In FIG. 36E, GM-CSF expressed in mammaliancells is first treated with sialidase to trim back sialic acid residues,then PEGylated using ST3Gal3 and a donor of PEG-sialic acid, and then isfurther sialylated using ST3Gal3 and a sialic acid donor. In FIG. 36F,GM-CSF expressed in mammalian cells is modified by capping appropriateterminal residues with a sialic acid donor that is modified withlevulinic acid, adding a reactive ketone to the sialic acid donor. Afteraddition to a glycosyl residue of the peptide, the ketone is derivatizedwith a moiety such as a hydrazine- or amine-PEG. In FIG. 36G, GM-CSFexpressed in mammalian cells is sialylated using a sialic acid donor andα2,8-sialyltransferase. In FIG. 36I, GM-CSF expressed in insect cells ismodified by addition of N-acetylglucosamine using a suitable donor andone or more of GnT-I, II, IV, and V, followed by addition of PEGylatedgalactose using a suitable donor and a galactosyltransferase. In FIG.36J, yeast expressed GM-CSF is first treated with endoglycanase and/ormannosidase to trim back the glycosyl units, and subsequently PEGylatedusing a galactosyltransferase and a donor of PEG-galactose. In FIG. 36K,GM-CSF expressed in mammalian cells is first treated with sialidase totrim back sialic acid residues, and is subsequently sialylated usingST3Gal3 and a sialic acid donor. The polypeptide is then contacted withST3 Gal1 and two reactive sialic acid residues connected via a linker,so that the polypeptide is attached to one reactive sialic acid via thelinker and second sialic acid residue. The polypeptide is furthercontacted with ST3Gal3 and transferrin, and thus becomes connected withtransferrin.

In another exemplary embodiment, the invention provides methods formodification of Interferon gamma (IFNγ). FIGS. 37A to 37N contain someexamples. In FIG. 37B, IFNγ expressed in a variety of mammalian cells isfirst treated with sialidase to trim back terminal sialic acid residues,and is subsequently PEGylated using ST3Gal3 and a donor of PEG-sialicacid. In FIG. 37C, IFNγ expressed in a mammalian system is first treatedwith sialidase to trim back terminal sialic acid residues. Thepolypeptide is then PEGylated using ST3Gal3 and a donor of PEG-sialicacid, and is further sialylated with ST3Gal3 and a donor of sialic acid.In FIG. 37D, mammalian cell expressed IFNγ is first treated withsialidase and α-galactosidase to trim back sialic acid and galactoseresidues. The polypeptide is then galactosylated using a galactose donorand a galactosyltransferase. Then, IFNγ is PEGylated using a donor ofPEG-sialic acid and ST3Gal3. In FIG. 37E, lFNγ that is expressed in amammalian system is first treated with sialidase to trim back terminalsialic acid residues. The polypeptide is then PEGylated using ST3Gal3and a donor of PEG-sialic acid, and is further sialylated with ST3Gal3and a sialic acid donor. FIG. 37F describes another method for modifyingIFNγ expressed in a mammalian system. The protein is modified by cappingappropriate terminal residues with a sialic acid donor that is modifiedwith levulinic acid, adding a reactive ketone to the sialic acid donor.After addition to a glycosyl residue of the peptide, the ketone isderivatized with a moiety such as a hydrazine- or amine-PEG. In FIG.37G, IFNγ expressed in mammalian cells is remodeled by addition ofsialic acid using a sialic acid donor and an α2,8-sialyltransferase. InFIG. 37I, IFNγ expressed in insect or fungal cells is modified byaddition of N-acetylglucosamine using an appropriate donor and one ormore of GnT-I, II, IV, and V. The protein is further modified byaddition of PEG moieties using a donor of PEGylated galactose and agalactosyltransferase. FIG. 37J offers a method for modifying IFNγexpressed in yeast. The polypeptide is first treated with endoglycanaseto trim back the saccharide chains, and then galactosylated using agalactose donor and a galactosyltransferase. Then, IFNγ is PEGylatedusing a donor of PEGylated sialic acid and ST3Gal3. In FIG. 37K, IFNγproduced by mammalian cells is modified as follows: the polypeptide isfirst contacted with ST3Gal3 and a donor of sialic acid that isderivatized with a reactive galactose via a linker, so that thepolypeptide is attached to the reactive galactose via the linker andsialic acid residue. The polypeptide is then contacted with agalactosyltransferase and transferrin pre-treated with endoglycanase,and thus becomes connected with transferrin via the galactose residue.In the scheme illustrated by FIG. 37L, IFNγ, which is expressed in amammalian system, is modified via the action of ST3Gal3: PEGylatedsialic acid is transferred from a suitable donor to IFNγ. FIG. 37M is anexample of modifying IFNγ expressed in insect or fungal cells, wherePEGylation of the polypeptide is achieved by transferring PEGylatedN-acetylglucosamine from a donor to IFNγ using GnT-I and/or II. In FIG.37N, IFNγ expressed in a mammalian system is remodeled with addition ofPEGylated sialic acid using a suitable donor and anα2,8-sialyltransferase.

In another exemplary embodiment, the invention provides methods formodifying α₁ anti-trypsin (α1-protease inhibitor). Some such examplescan be found in FIGS. 38A to 38N. In FIG. 38B, a, anti-trypsin expressedin a variety of mammalian cells is first treated with sialidase to trimback sialic acid residues. PEGylated sialic acid residues are then addedusing an appropriate donor, such as CMP-SA-PEG, and a sialyltransferase,such as ST3Gal3. FIG. 38C demonstrates another scheme of α₁ anti-trypsinmodification. α₁ anti-trypsin expressed in a mammalian system is firsttreated with sialidase to trim back sialic acid residues. Sialic acidresidues derivatized with PEG are then added using an appropriate donorand a sialyltransferase, such as ST3Gal3. Subsequently, the molecule isfurther modified by the addition of sialic acid residues using a sialicacid donor and ST3Gal3. Optionally, mammalian cell expressed α₁anti-trypsin is first treated with sialidase and α-galactosidase to trimback terminal sialic acid and α-linkage galactose residues. Thepolypeptide is then galactosylated using galactosyltransferase and asuitable galactose donor. Further, sialic acid derivatized with PEG isadded by the action of ST3Gal3 using a PEGylated sialic acid donor. InFIG. 38D, α₁ anti-trypsin expressed in a mammalian system first has theterminal sialic acid residues trimmed back using sialidase. PEG is thenadded to N-linked glycosyl residues via the action of ST3Gal3, whichmediates the transfer of PEGylated sialic acid from a donor, such asCMP-SA-PEG, to α₁ anti-trypsin. More sialic acid residues aresubsequently attached using a sialic acid donor and ST3Gal3. FIG. 38Eillustrates another process through which α₁ anti-trypsin is remodeled.α₁ anti-trypsin expressed in mammalian cells is modified by cappingappropriate terminal residues with a sialic acid donor that is modifiedwith levulinic acid, adding a reactive ketone to the sialic acid donor.After addition to a glycosyl residue of the peptide, the ketone isderivatized with a moiety such as a hydrazine- or amine-PEG. In FIG.38F, yet another method of α₁ anti-trypsin modification is disclosed. α₁anti-trypsin obtained from a mammalian expression system is remodeledwith addition of sialic acid using a sialic acid donor and anα2,8-sialyltransferase. In FIG. 38H, α₁ anti-trypsin is expressed ininsect or yeast cells, and remodeled by the addition of terminalN-acetylglucosamine residues by way of contacting the polypeptide withUDP-N-acetylglucosamine and one or more of GnT-I, II, IV, or V. Then,the polypeptide is modified with PEG moieties using a donor of PEGylatedgalactose and a galactosyltransferase. In FIG. 38I, α₁ anti-trypsinexpressed in yeast cells is treated first with endoglycanase to trimback glycosyl chains. It is then galactosylated with agalactosyltransferase and a galactose donor. Then, the polypeptide isPEGylated using ST3Gal3 and a donor of PEG-sialic acid. In FIG. 38J, α₁anti-trypsin is expressed in a mammalian system. The polypeptide isfirst contacted with ST3Gal3 and a donor of sialic acid that isderivatized with a reactive galactose via a linker, so that thepolypeptide is attached to the reactive galactose via the linker andsialic acid residue. The polypeptide is then contacted with agalactosyltransferase and transferrin pre-treated with endoglycanase,and thus becomes connected with transferrin via the galactose residue.In FIG. 38L, α₁ anti-trypsin expressed in yeast is first treated withendoglycanase to trim back its glycosyl groups. The protein is thenPEGylated using a galactosyltransferase and a donor of galactose with aPEG moiety. In FIG. 38M, α₁ anti-trypsin expressed in plant cells istreated with hexosaminidase, mannosidase, and xylosidase to trim backits glycosyl chains, and subsequently modified with N-acetylglucosaminederivatized with a PEG moiety, using N-acetylglucosamine transferase anda suitable donor. In FIG. 38N, a, anti-trypsin expressed in mammaliancells is modified by adding PEGylated sialic acid residues using ST3Gal3and a donor of sialic acid derivatized with PEG.

In another exemplary embodiment, the invention provides methods formodifying glucocerebrosidase (β-glucosidase, Cerezyme™ or Ceredase™), asshown in FIGS. 39A to 39K. In FIG. 39B, Cerezyme™ expressed in amammalian system is first treated with sialidase to trim back terminalsialic acid residues, and is then PEGylated using ST3Gal3 and a donor ofPEG-sialic acid. In FIG. 39C, Cerezyme™ expressed in mammalian cells isfirst treated with sialidase to trim back the sialic acid residues, thenhas mannose-6-phosphate group attached using ST3Gal3 and a reactivesialic acid derivatized with mannose-6-phosphate, and then is sialylatedusing ST3Gal3 and a sialic acid donor. Optionally, NSO cell expressedCerezyme™ is first treated with sialidase and galactosidase to trim backthe glycosyl groups, and is then galactosylated using a galactose donorand an α-galactosyltransferase. Then, mannose-6-phosphate moiety isadded to the molecule using ST3Gal3 and a reactive sialic acidderivatized with mannose-6-phosphate. In FIG. 39D, Cerezyme™ expressedin mammalian cells is first treated with sialidase to trim back thesialic acid residues, it is then PEGylated using ST3Gal3 and a donor ofPEG-sialic acid, and is then sialylated using ST3Gal3 and a sialic aciddonor. In FIG. 39E, Cerezyme™ expressed in mammalian cells is modifiedby capping appropriate terminal residues with a sialic acid donor thatis modified with levulinic acid, adding a reactive ketone to the sialicacid donor. After addition to a glycosyl residue of the peptide, theketone is derivatized with a moiety such as one or moremannose-6-phosphate groups. In FIG. 39F, Cerezyme™ expressed inmammalian cells is sialylated using a sialic acid donor andα2,8-sialyltransferase. In FIG. 39H, Cerezyme™ expressed in insect cellsfirst has N-acetylglucosamine added using a suitable donor and one ormore of GnT-I, II, IV, and V, and then is PEGylated using agalactosyltransferase and a donor of PEG-galactose. In FIG. 39I,Cerezyrme™ expressed in yeast is first treated with endoglycanase totrim back the glycosyl groups, then galactosylated using a galactosedonor and a galactosyltransferase, and then PEGylated using ST3Gal3 anda donor of PEG-sialic acid. In FIG. 39JK, Cerezyme™ expressed inmammalian cells is first contacted with ST3Gal3 and two reactive sialicacid residues connected via a linker, so that the polypeptide isattached to one reactive sialic acid via the linker and the secondsialic acid residue. The polypeptide is then contacted with ST3Gal3 anddesialylated transferrin, and thus becomes connected with transferrin.Then, the polypeptide is sialylated using a sialic acid donor andST3Gal3.

In another exemplary embodiment, the invention provides methods formodifying Tissue-Type Plasminogen Activator (TPA) and its mutant.Several specific modification schemes are presented in FIGS. 40A to 40W.FIG. 40B illustrates one modification procedure: after TPA is expressedby mammalian cells, it is treated with one or more of mannosidase(s) andsialidase to trim back mannosyl and/or sialic acid residues. TerminalN-acetylglucosamine is then added by contacting the polypeptide with asuitable donor of N-acetylglucosamine and one or more of GnT-I, II, IV,and V. TPA is further galactosylated using a galactose donor and agalactosyltransferase. Then, PEG is attached to the molecule by way ofsialylation catalyzed by ST3Gal3 and using a donor of sialic acidderivatized with a PEG moiety. In FIG. 40C, TPA is expressed in insector fungal cells. The modification includes the steps of addition ofN-acetylglucosamine using an appropriate donor of N-acetylglucosamineand GnT-I and/or II; galactosylation using a galactose donor and agalactosyltransferase; and attachment of PEG by way of sialylation usingST3Gal3 and a donor of sialic acid derivatized with PEG. In FIG. 40D,TPA is expressed in yeast and subsequently treated with endoglycanase totrim back the saccharide chains. The polypeptide is further PEGylatedvia the action of a galactosyltransferase, which catalyzes the transferof a PEG-galactose from a donor to TPA. In FIG. 40E, TPA is expressed ininsect or yeast cells. The polypeptide is then treated with α- andβ-mannosidases to trim back terminal mannosyl residues. Further, PEGmoieties are attached to the molecule via transfer of PEG-galactose froma suitable donor to TPA, which is mediated by a galactosyltransferase.FIG. 40F provides a different method for modification of TPA obtainedfrom an insect or yeast system: the polypeptide is remodeled by additionof N-acetylglucosamine using a donor of N-acetylglucosamine and GnT-Iand/or II, followed by PEGylation using a galactosyltransferase and adonor of PEGylated galactose. FIG. 40G offers another scheme forremodeling TPA expressed in insect or yeast cells. TerminalN-acetylglucosamine is added using a donor of N-acetylglucosamine andGnT-I and/or II. A galactosidase that is modified to operate in asynthetic, rather than a hydrolytic manner, is utilized to add PEGylatedgalactose from a proper donor to the N-acetylglucosamine residues. InFIG. 40I, TPA expressed in a mammalian system is first treated withsialidase and galactosidase to trim back sialic acid and galactoseresidues. The polypeptide is further modified by capping appropriateterminal residues with a sialic acid donor that is modified withlevulinic acid, adding a reactive ketone to the sialic acid donor. Afteraddition to a glycosyl residue of the peptide, the ketone is derivatizedwith a moiety such as a hydrazine- or amine-PEG. In FIG. 40J, TPA, whichis expressed in a mammalian system, is remodeled following this scheme:first, the polypeptide is treated with α- and β-mannosidases to trimback the terminal mannosyl residues; sialic acid residues are thenattached to terminal galactosyl residues using a sialic acid donor andST3Gal3; further, TPA is PEGylated via the transfer of PEGylatedgalactose from a donor to a N-acetylglucosaminyl residue catalyzed by agalactosyltransferase. In FIG. 40K, TPA is expressed in a plant system.The modification procedure in this example is as follows: TPA is firsttreated with hexosaminidase, mannosidase, and xylosidase to trim backits glycosyl groups; PEGylated N-acetylglucosamine is then added to TPAusing a proper donor and N-acetylglucosamine transferase. In FIG. 40M, aTPA mutant (TNK TPA), expressed in mammalian cells, is remodeled.Terminal sialic acid residues are first trimmed back using sialidase;ST3Gal3 is then used to transfer PEGylated sialic acid from a donor toTNK TPA, such that the polypeptide is PEGylated. In FIG. 40N, TNK TPAexpressed in a mammalian system is first treated with sialidase to trimback terminal sialic acid residues. The protein is then PEGylated usingCMP-SA-PEG as a donor and ST3Gal3, and further sialylated using a sialicacid donor and ST3Gal3. In FIG. 40O, NSO cell expressed TNK TPA is firsttreated with sialidase and α-galactosidase to trim back terminal sialicacid and galactose residues. TNK TPA is then galactosylated using agalactose donor and a galactosyltransferase. The last step in thisremodeling scheme is transfer of sialic acid derivatized with PEG moietyfrom a donor to TNK TPA using a sialyltransferase such as ST3Gal3. InFIG. 40Q, TNK TPA is expressed in a mammalian system and is firsttreated with sialidase to trim back terminal sialic acid residues. Theprotein is then PEGylated using ST3Gal3 and a donor of PEGylated sialicacid. Then, the protein is sialylated using a sialic acid donor andST3Gal3. In FIG. 40R, TNK TPA expressed in a mammalian system ismodified by capping appropriate terminal residues with a sialic aciddonor that is modified with levulinic acid, adding a reactive ketone tothe sialic acid donor. After addition to a glycosyl residue of thepeptide, the ketone is derivatized with a moiety such as a hydrazine- oramine-PEG. In FIG. 40S, TNK TPA expressed in mammalian cells is modifiedvia a different method: the polypeptide is remodeled with addition ofsialic acid using a sialic acid donor and α2,8-sialyltransferase. InFIG. 40U, TNK TPA expressed in insect cells is remodeled by addition ofN-acetylglucosamine using an appropriate donor and one or more of GnT-I,II, IV, and V. The protein is further modified by addition of PEGmoieties using a donor of PEGylated galactose and agalactosyltransferase. In FIG. 40V, TNK TPA is expressed in yeast. Thepolypeptide is first treated with endoglycanase to trim back itsglycosyl chains and then PEGylated using a galactose donor derivatizedwith PEG and a galactosyltransferase. In FIG. 40W, TNK TPA is producedin a mammalian system. The polypeptide is first contacted with ST3Gal3and a donor of sialic acid that is derivatized with a reactive galactosevia a linker, so that the polypeptide is attached to the reactivegalactose via the linker and sialic acid residue. The polypeptide isthen contacted with a galactosyltransferase and anti-TNF IG chimeraproduced in CHO, and thus becomes connected with the chimera via thegalactose residue.

In another exemplary embodiment, the invention provides methods formodifying Interleukin-2 (IL-2). FIGS. 41A to 41G provide some examples.FIG. 41B provides a two-step modification scheme: IL-2 produced bymammalian cells is first treated with sialidase to trim back itsterminal sialic acid residues, and is then PEGylated using ST3Gal3 and adonor of PEGylated sialic acid. In FIG. 41C, insect cell expressed IL-2is modified first by galactosylation using a galactose donor and agalactosyltransferase. Subsequently, IL-2 is PEGylated using ST3Gal3 anda donor of PEGylated sialic acid. In FIG. 41D, IL-2 expressed inbacteria is modified with N-acetylgalactosamine using a proper donor andN-acetylgalactosamine transferase, followed by a step of PEGylation witha PEG-sialic acid donor and a sialyltransferase. FIG. 41E offers anotherscheme of modifying IL-2 produced by a mammalian system. The polypeptideis modified by capping appropriate terminal residues with a sialic aciddonor that is modified with levulinic acid, adding a reactive ketone tothe sialic acid donor. After addition to a glycosyl residue of thepeptide, the ketone is derivatized with a moiety such as a hydrazine- oramine-PEG. FIG. 41F illustrates an example of remodeling IL-2 expressedby E. coli. The polypeptide is PEGylated using a reactiveN-acetylgalactosamine complex derivatized with a PEG group and an enzymethat is modified so that it functions as a synthetic enzyme rather thana hydrolytic one. In FIG. 41G, IL-2 expressed by bacteria is modified byaddition of PEGylated N-acetylgalactosamine using a proper donor andN-acetylgalactosamine transferase.

In another exemplary embodiment, the invention provides methods formodifying Factor VIII, as shown in FIGS. 42A to 42N. In FIG. 42B, FactorVIII expressed in mammalian cells is first treated with sialidase totrim back the sialic acid residues, and is then PEGylated using ST3Gal3and a donor of PEG-sialic acid. In FIG. 42C, Factor VIII expressed inmammalian cells is first treated with sialidase to trim back the sialicacid residues, then PEGylated using ST3Gal3 and a proper donor, and isthen further sialylated using ST3Gal1 and a sialic acid donor.

In FIG. 42E, mammalian cell produced Factor VIII is modified by thesingle step of PEGylation, using ST3Gal3 and a donor of PEGylated sialicacid. FIG. 42F offers another example of modification of Factor VIIIthat is expressed by mammalian cells. The protein is PEGylated usingST3Gal1 and a donor of PEGylated sialic acid. In FIG. 42G, mammaliancell expressed Factor VIII is remodeled following another scheme: it isPEGylated using α2,8-sialyltransferase and a donor of PEG-sialic acid.In FIG. 42I, Factor VIII produce by mammalian cells is modified bycapping appropriate terminal residues with a sialic acid donor that ismodified with levulinic acid, adding a reactive ketone to the sialicacid donor. After addition to a glycosyl residue of the peptide, theketone is derivatized with a moiety such as a hydrazine- or amine-PEG.In FIG. 42J, Factor VIII expressed by mammalian cells is first treatedwith Endo-H to trim back glycosyl groups. It is then PEGylated using agalactosyltransferase and a donor of PEG-galactose. In FIG. 42K, FactorVIII expressed in a mammalian system is first sialylated using ST3Gal3and a sialic acid donor, then treated with Endo-H to trim back theglycosyl groups, and then PEGylated with a galactosyltransferase and adonor of PEG-galactose. In FIG. 42L, Factor VIII expressed in amammalian system is first treated with mannosidases to trim backterminal mannosyl residues, then has an N-acetylglucosamine group addedusing a suitable donor and GnT-I and/or II, and then is PEGylated usinga galactosyltransferase and a donor of PEG-galactose. In FIG. 42M,Factor VIII expressed in mammalian cells is first treated withmannosidases to trim back mannosyl units, then has N-acetylglucosaminegroup added using N-acetylglucosamine transferase and a suitable donor.It is further galactosylated using a galactosyltransferase and agalactose donor, and then sialylated using ST3Gal3 and a sialic aciddonor. In FIG. 42N, Factor VIII is produced by mammalian cells andmodified as follows: it is first treated with mannosidases to trim backthe terminal mannosyl groups. A PEGylated N-acetylglucosamine group isthen added using GnT-I and a suitable donor of PEGylatedN-acetylglucosamine.

In another exemplary embodiment, the invention provides methods formodifying urokinase, as shown in FIGS. 43A to 43M. In FIG. 43B,urokinase expressed in mammalian cells is first treated with sialidaseto trim back sialic acid residues, and is then PEGylated using ST3Gal3and a donor of PEGylated sialic acid. In FIG. 43C, urokinase expressedin mammalian cells is first treated with sialidase to trim back sialicacid residues, then PEGylated using ST3Gal3 and a donor of PEGylatedsialic acid, and then sialylated using ST3Gal3 and a sialic acid donor.Optionally, urokinase expressed in a mammalian system is first treatedwith sialidase and galactosidase to trim back glycosyl chains, thengalactosylated using a galactose donor and an α-galactosyltransferase,and then PEGylated using ST3Gal3 or sialyltransferase and a donor ofPEG-sialic acid. In FIG. 43D, urokinase expressed in mammalian cells isfirst treated with sialidase to trim back sialic acid residues, thenPEGylated using ST3Gal3 and a donor of PEG-sialic acid, and then furthersialylated using ST3Gal3 and a sialic acid donor. In FIG. 43E, urokinaseexpressed in mammalian cells is modified by capping appropriate terminalresidues with a sialic acid donor that is modified with levulinic acid,adding a reactive ketone to the sialic acid donor. After addition to aglycosyl residue of the peptide, the ketone is derivatized with a moietysuch as a hydrazine- or amine-PEG. In FIG. 43F, urokinase expressed inmammalian cells is sialylated using a sialic acid donor andα2,8-sialyltransferase. In FIG. 43H, urokinase expressed in insect cellsis modified in the following steps: first, N-acetylglucosamine is addedto the polypeptide using a suitable donor of N-acetylglucosamine and oneor more of GnT-I, II, IV, and V; then PEGylated galactose is added,using a galactosyltransferase and a donor of PEG-galactose. In FIG. 43I,urokinase expressed in yeast is first treated with endoglycanase to trimback glycosyl groups, then galactosylated using a galactose donor and agalactosyltransferase, and then PEGylated using ST3Gal3 and a donor ofPEG-sialic acid. In FIG. 43J, urokinase expressed in mammalian cells isfirst contacted with ST3Gal3 and two reactive sialic acid residues thatare connected via a linker, so that the polypeptide is attached to onereactive sialic acid via the linker and second sialic acid residue. Thepolypeptide is then contacted with ST3Gal1 and desialylated urokinaseproduced in mammalian cells, and thus becomes connected with a secondmolecule of urokinase. Then, the whole molecule is further sialylatedusing a sialic donor and ST3Gal1 and/or ST3Gal3. In FIG. 43K, isolatedurokinase is first treated with sulfohydrolase to remove sulfate groups,and is then PEGylated using a sialyltransferase and a donor ofPEG-sialic acid. In FIG. 43LM, isolated urokinase is first treated withsulfohydrolase and hexosaminidase to remove sulfate groups andhexosamine groups, and then PEGylated using a galactosyltransferase anda donor of PEG-galactose.

In another exemplary embodiment, the invention provides methods formodifing DNase I, as shown in FIGS. 44A to 44J. In FIG. 44B, DNase I isexpressed in a mammalian system and modified in the following steps:first, the protein is treated with sialidase to trim back the sialicacid residues; then the protein is PEGylated with ST3Gal3 using a donorof PEG-sialic acid. In FIG. 44C, DNase I expressed in mammalian cells isfirst treated with sialidase to trim back the sialic acid residues, thenPEGylated with ST3Gal3 using a PEG-sialic acid donor, and is thensialylated using ST3Gal3 and a sialic acid donor. Optionally, DNase Iexpressed in a mammalian system is first exposed to sialidase andgalactosidase to trim back the glycosyl groups, then galactosylatedusing a galactose donor and an α-galactosyltransferase, and thenPEGylated using ST3Gal3 or sialyltransferase and a donor of PEG-sialicacid. In FIG. 44D, DNase I expressed in mammalian cells is first treatedwith sialidase to trim back the sialic acid residues, then PEGylatedusing ST3Gal3 and a PEG-sialic acid donor, and then sialylated withST3Gal3 using a sialic acid donor. In FIG. 44E, DNase I expressed inmammalian cells is modified by capping appropriate terminal residueswith a sialic acid donor that is modified with levulinic acid, adding areactive ketone to the sialic acid donor. After addition to a glycosylresidue of the peptide, the ketone is derivatized with a moiety such asa hydrazine- or amine-PEG. In FIG. 44F, DNase I expressed in mammaliancells is sialylated using a sialic acid donor andα2,8-sialyltransferase. In FIG. 44H, DNase I expressed in insect cellsfirst has N-acetylglucosamine added using a suitable donor and one ormore of GnT-I, II, IV, and V. The protein is then PEGylated using agalactosyltransferase and a donor of PEG-galactose. In FIG. 44I, DNase Iexpressed in yeast is first treated with endoglycanase to trim back theglycosyl units, then galactosylated using a galactose donor and agalactosyltransferase, and then PEGylated using ST3Gal3 and a donor ofPEG-sialic acid. In FIG. 44JK, DNase I expressed in mammalian cells isfirst contacted with ST3Gal3 and two reactive sialic acid residuesconnected via a linker, so that the polypeptide is attached to onereactive sialic acid via the linker and the second sialic acid residue.The polypeptide is then contacted with ST3Gal1 and desialylatedα-1-protease inhibitor, and thus becomes connected with the inhibitorvia the sialic acid residue. Then, the polypeptide is further sialylatedusing a suitable donor and ST3Gal1 and/or ST3Gal3.

In another exemplary embodiment, the invention provides methods formodifying insulin that is mutated to contain an N-glycosylation site, asshown in FIGS. 45A to 45L. In FIG. 45B, insulin expressed in a mammaliansystem is first treated with sialidase to trim back the sialic acidresidues, and then PEGylated using ST3Gal3 and a PEG-sialic acid donor.In FIG. 45C, insulin expressed in insect cells is modified by additionof PEGylated N-acetylglucosamine using an appropriate donor and GnT-Iand/or II. In FIG. 45D, insulin expressed in yeast is first treated withEndo-H to trim back the glycosyl groups, and then PEGylated using agalactosyltransferase and a donor of PEG-galactose. In FIG. 45F, insulinexpressed in mammalian cells is first treated with sialidase to trimback the sialic acid residues and then PEGylated using ST3Gal1 and adonor of PEG-sialic acid. In FIG. 45G, insulin expressed in insect cellsis modified by means of addition of PEGylated galactose using a suitabledonor and a galactosyltransferase. In FIG. 45H, insulin expressed inbacteria first has N-acetylgalactosamine added using a proper donor andN-acetylgalactosamine transferase. The polypeptide is then PEGylatedusing a sialyltransferase and a donor of PEG-sialic acid. In FIG. 45J,insulin expressed in bacteria is modified through a different method:PEGylated N-acetylgalactosamine is added to the protein using a suitabledonor and N-acetylgalactosamine transferase. In FIG. 45K, insulinexpressed in bacteria is modified following another scheme: thepolypeptide is first contacted with N-acetylgalactosamine transferaseand a reactive N-acetylgalactosamine that is derivatized with a reactivesialic acid via a linker, so that the polypeptide is attached to thereactive sialic acid via the linker and N-acetylgalactosamine. Thepolypeptide is then contacted with ST3Gal3 and asialo-transferrin, andtherefore becomes connected with transferrin. Then, the polypeptide issialylated using ST3Gal3 and a sialic acid donor. In FIG. 45L, insulinexpressed in bacteria is modified using yet another method: thepolypeptide is first exposed to NHS—CO-linker-SA-CMP and becomesconnected to the reactive sialic acid residue via the linker. Thepolypeptide is then conjugated to transferrin using ST3Gal3 andasialo-transferrin. Then, the polypeptide is further sialylated usingST3Gal3 and a sialic acid donor.

In another exemplary embodiment, the invention provides methods formodifying Hepatitis B antigen (M antigen-preS2 and S), as shown in FIGS.46A to 46K. In FIG. 46B, M-antigen is expressed in a mammalian systemand modified by initial treatment of sialidase to trim back the sialicacid residues and subsequent conjugation with lipid A, using ST3Gal3 anda reactive sialic acid linked to lipid A via a linker. In FIG. 46C,M-antigen expressed in mammalian cells is first treated with sialidaseto trim back the terminal sialic acid residues, then conjugated withtetanus toxin via a linker using ST3Gal1 and a reactive sialic acidresidue linked to the toxin via the linker, and then sialylated usingST3Gal3 and a sialic acid donor. In FIG. 46D, M-antigen expressed in amammalian system is first treated with a galactosidase to trim backgalactosyl residues, and then sialylated using ST3Gal3 and a sialic aciddonor. The polypeptide then has sialic acid derivatized with KLH addedusing ST3Gal1 and a suitable donor. In FIG. 46E, yeast expressedM-antigen is first treated with a mannosidase to trim back the mannosylresidues, and then conjugated to a diphtheria toxin using GnT-I and adonor of N-acetylglucosamine linked to the diphtheria toxin. In FIG.46F, mammalian cell expressed M-antigen is modified by cappingappropriate terminal residues with a sialic acid donor that is modifiedwith levulinic acid, adding a reactive ketone to the sialic acid donor.After addition to a glycosyl residue of the peptide, the ketone isderivatized with a moiety such as a hydrazine- or amine-PEG. In FIG.46G, M-antigen obtained from a mammalian system is remodeled bysialylation using a sialic acid donor and poly α2,8-sialyltransferase.In FIG. 46I, M-antigen expressed in insect cells is conjugated to aNeisseria protein by using GnT-II and a suitable donor ofN-acetylglucosamine linked to the Neisseria protein. In FIG. 46J, yeastexpressed M-antigen is first treated with endoglycanase to trim back itsglycosyl chains, and then conjugated to a Neisseria protein using agalactosyltransferase and a proper donor of galactose linked to theNeisseria protein. FIG. 46K is another example of modification ofM-antigen expressed in yeast. The polypeptide is first treated withmannosidases to trim back terminal mannosyl residues, and then hasN-acetylglucosamine added using GnT-I and/or II. Subsequently, thepolypeptide is galactosylated using a galactose donor and agalactosyltransferase, and then capped with sialic acid residues using asialyltransferase and a sialic acid donor.

In another exemplary embodiment, the invention provides methods formodifying human growth hormone (N, V, and variants thereof), as shown inFIGS. 47A to 47K. In FIG. 47B, human growth hormone either mutated tocontain a N-linked site, or a naturally occurring isoform that has anN-linked side (i.e., the placental enzyme) produced by mammalian cellsis first treated with sialidase to trim back terminal sialic acidresidues and subsequently PEGylated with ST3Gal3 and using a donor ofPEGylated sialic acid. In FIG. 47C, human growth hormone expressed ininsect cells is modified by addition of PEGylated N-acetylglucosamineusing GnT-I and/or II and a proper donor of PEGylatedN-acetylglucosamine. In FIG. 47D, human growth hormone is expressed inyeast, treated with Endo-H to trim back glycosyl groups, and furtherPEGylated with a galactosyltransferase using a donor of PEGylatedgalactose. In FIG. 47F, human growth hormone-mucin fusion proteinexpressed in a mammalian system is modified by initial treatment ofsialidase to trim back sialic acid residues and subsequent PEGylationusing a donor of PEG-sialic acid and ST3Gall. In FIG. 47G, human growthhormone-mucin fusion protein expressed in insect cells is remodeled byPEGylation with a galactosyltransferase and using a donor of PEGylatedgalactose. In FIG. 47H, human growth hormone-mucin fusion protein isproduced in bacteria. N-acetylgalactosamine is first added to the fusionprotein by the action of N-acetylgalactosamine transferase using a donorof N-acetylgalactosamine, followed by PEGylation of the fusion proteinusing a donor of PEG-sialic acid and a sialyltransferase. FIG. 47Idescribes another scheme of modifying bacterially expressed human growthhormone-mucin fusion protein: the fusion protein is PEGylated throughthe action of N-acetylgalactosamine transferase using a donor ofPEGylated N-acetylgalactosamine. FIG. 47J provides a further remodelingscheme for human growth hormone-mucin fusion protein. The fusion proteinis first contacted with N-acetylgalactosamine transferase and a donor ofN-acetylgalactosamine that is derivatized with a reactive sialic acidvia a linker, so that the fusion protein is attached to the reactivesialic acid via the linker and N-acetylgalactosamine. The fusion proteinis then contacted with a sialyltransferase and asialo-transferrin, andthus becomes connected with transferrin via the sialic acid residue.Then, the fusion protein is capped with sialic acid residues usingST3Gal3 and a sialic acid donor. In FIG. 47K, yet another scheme isgiven for modification of human growth hormone(N) produced in bacteria.The polypeptide is first contacted with NHS—CO-linker-SA-CMP and becomescoupled with the reactive sialic acid through the linker. Thepolypeptide is then contacted with ST3Gal3 and asialo-transferrin andbecomes linked to transferrin via the sialic acid residue. Then, thepolypeptide is sialylated using ST3Gal3 and a sialic acid donor.

In another exemplary embodiment, the invention provides methods forremodeling TNF receptor IgG fusion protein (TNFR-IgG, or Enbre™), asshown in FIGS. 48A to 48G. FIG. 48B illustrates a modification procedurein which TNFR-IgG, expressed in a mammalian system is first sialylatedwith a sialic acid donor and a sialyltransferase, ST3Gal1; the fusionprotein is then galactosylated with a galactose donor and agalactosyltransferase; then, the fusion protein is PEGylated via theaction of ST3Gal3 and a donor of sialic acid derivatized with PEG. InFIG. 48C, TNFR-IgG expressed in mammalian cells is initially treatedwith sialidase to trim back sialic acid residues. PEG moieties aresubsequently attached to TNFR-IgG by way of transferring PEGylatedsialic acid from a donor to the fusion protein in a reaction catalyzedby ST3Gall. In FIG. 48D, TNFR-IgG is expressed in a mammalian system andmodified by addition of PEG through the galactosylation process, whichis mediated by a galactosyltransferase using a PEG-galactose donor. InFIG. 48E, TNFR-IgG is expressed in a mammalian system. The first step inremodeling of the fusion protein is adding O-linked sialic acid residuesusing a sialic acid donor and a sialyltransferase, ST3Gal1.Subsequently, PEGylated galactose is added to the fusion protein using agalactosyltransferase and a suitable donor of galactose with a PEGmoiety. In FIG. 48F, TNFR-IgG expressed in mammalian cells is modifiedfirst by capping appropriate terminal residues with a sialic acid donorthat is modified with levulinic acid, adding a reactive ketone to thesialic acid donor. After addition to a glycosyl residue of the fusionprotein, the ketone is derivatized with a moiety such as a hydrazine- oramine-PEG. In FIG. 48G, TNFR-IgG expressed in mammalian cells isremodeled by 2,8-sialyltransferase, which catalyzes the reaction inwhich PEGylated sialic acid is transferred to the fusion protein from adonor of sialic acid with a PEG moiety.

In another exemplary embodiment, the invention provides methods forgenerating Herceptin™ conjugates, as shown in FIGS. 49A to 49D. In FIG.49B, Herceptin™ is expressed in a mammalian system and is firstgalactosylated using a galactose donor and a galactosyltransferase.Herceptin™ is then conjugated with a toxin via a sialic acid through theaction of ST3Gal3 using a reactive sialic acid-toxin complex. In FIG.49C, Herceptin™ produced in either mammalian cells or fungi isconjugated to a toxin through the process of galactosylation, using agalactosyltransferase and a reactive galactose-toxin complex. FIG. 49Dcontains another scheme of making Herceptin™ conjugates: Herceptin™produced in fungi is first treated with Endo-H to trim back glycosylgroups, then galactosylated using a galactose donor and agalactosyltransferase, and then conjugated with a radioisotope by way ofsialylation, by using ST3Gal3 and a reactive sialic acid-radioisotopecomplex. Alternatively, the reactive sialic acid moiety may haveattached only the chelating moiety can then be loaded with radioisotopeat a subsequent stage.

In another exemplary embodiment, the invention provides methods formaking Synagis™ conjugates, as shown in FIGS. 50A to 50D. In FIG. 50B,Synagis™ expressed in mammalian cells is first galactosylated using agalactose donor and a galactosyltransferase, and then PEGylated usingST3Gal3 and a donor of PEG-sialic acid. In FIG. 50C, Synagis™ expressedin mammalian or fungal cells is PEGylated using a galactosyltransferaseand a donor of PEG-galactose. In FIG. 50D, Synagis™ expressed in firsttreated with Endo-H to trim back the glycosyl groups, thengalactosylated using a galactose donor and a galactosyltransferase, andis then PEGylated using ST3Gal3 and a donor of PEG-sialic acid.

In another exemplary embodiment, the invention provides methods forgenerating Remicade™ conjugates, as shown in FIGS. 51A to 51D. In FIG.5B, Remicade™ expressed in a mammalian system is first galactosylatedusing a galactose donor and a galactosyltransferase, and then PEGylatedusing ST3Gal3 and a donor of PEG-sialic acid. In FIG. 51C, Remicade™expressed in a mammalian system is modified by addition of PEGylatedgalactose using a suitable donor and a galactosyltransferase. In FIG.51D, Remicade™ expressed in fungi is first treated with Endo-H to trimback the glycosyl chains, then galactosylated using a galactose donorand a galactosyltransferase, and then conjugated to a radioisotope usingST3Gal3 and a reactive sialic acid derivatized with the radioisotope.

In another exemplary embodiment, the invention provides methods formodifying Reopro, which is mutated to contain an N glycosylation site.FIGS. 52A to 52L contain such examples. In FIG. 52B, Reopro expressed ina mammalian system is first treated with sialidase to trim back thesialic acid residues, and then PEGylated using ST3Gal3 and a donor ofPEG-sialic acid. In FIG. 52C, Reopro expressed in insect cells ismodified by addition of PEGylated N-acetylglucosamine using anappropriate donor and GnT-I and/or II. In FIG. 52D, Reopro expressed inyeast is first treated with Endo-H to trim back the glycosyl groups.Subsequently, the protein is PEGylated using a galactosyltransferase anda donor of PEG-galactose. In FIG. 52F, Reopro expressed in mammaliancells is first treated with sialidase to trim back the sialic acidresidues and then PEGylated with ST3Gal1 using a donor of PEGylatedsialic acid. In FIG. 52G, Reopro expressed in insect cells is modifiedby PEGylation using a galactosyltransferase and a donor ofPEG-galactose. In FIG. 52H, Reopro expressed in bacterial first hasN-acetylgalactosamine added using N-acetylgalactosamine transferase anda suitable donor. The protein is then PEGylated using asialyltransferase and a donor of PEG-sialic acid. In FIG. 52J, Reoproexpressed in bacteria is modified in a different scheme: it is PEGylatedvia the action of N-acetylgalactosamine transferase, using a donor ofPEGylated N-acetylgalactosamine. In FIG. 52K, bacterially expressedReopro is modified in yet another method: first, the polypeptide iscontacted with N-acetylgalactosamine transferase and a donor ofN-acetylgalactosamine that is derivatized with a reactive sialic acidvia a linker, so that the polypeptide is attached to the reactive sialicacid via the linker and N-acetylgalactosamine. The polypeptide is thencontacted with ST3Gal3 and asialo-transferrin and thus becomes connectedwith transferrin via the sialic acid residue. Then, the polypeptide iscapped with sialic acid residues using a proper donor and ST3Gal3. FIG.52L offers an additional scheme of modifying bacterially expressedReopro. The polypeptide is first exposed to NHS—CO-linker-SA-CMP andbecomes connected with the reactive sialic acid through the linker. Thepolypeptide is then contacted with ST3Gal3 and asialo-transferrin andthus becomes connected with transferrin via the sialic acid residue.Then, the polypeptide is capped with sialic acid residues using a properdonor and ST3Gal3.

In another exemplary embodiment, the invention provides methods forproducing Rituxan™ conjugates. FIGS. 53A to 53G presents some examples.In FIG. 53B, Rituxan™ expressed in various mammalian systems is firstgalactosylated using a proper galactose donor and agalactosyltransferase. The peptide is then functionalized with a sialicacid derivatized with a toxin moiety, using a sialic acid donor andST3Gal3. In FIG. 53C, Rituxan™ expressed in mammalian cells or fungalcells is galactosylated using a galactosyltransferase and a galactosedonor, which provides the peptide galactose containing a drug moiety.FIG. 53D provides another example of remodeling Rituxan™ expressed in afungal system. The polypeptide's glycosyl groups are first trimmed backusing Endo-H. Galactose is then added using a galactosyltransferase anda galactose donor. Subsequently, a radioisotope is conjugated to themolecule through a radioisotope-complexed sialic acid donor and asialyltransferase, ST3Gal3. In FIG. 53F, Rituxan™ is expressed in amammalian system and first galactosylated using a galactosyltransferaseand a proper galactose donor; sialic acid with a PEG moiety is thenattached to the molecule using ST3Gal3 and a PEGylated sialic aciddonor. As shown in FIG. 53G, Rituxan™ expressed in fungi, yeast, ormammalian cells can also be modified in the following process: first,the polypeptide is treated with α- and β-mannosidases to remove terminalmannosyl residues; GlcNAc is then attached to the molecule using GnT-I,II and a GlcNAc donor, radioisotope is then attached by way ofgalactosylation using a galactosyltransferase and a donor of galactosethat is coupled to a chelating moiety capable of binding a radioisotope.

In another exemplary embodiment, the invention provides methods formodifying anti-thrombin III (AT III). FIGS. 54A to 54O present someexamples. In FIG. 54B, anti-thrombin III expressed in various mammaliansystems is remodeled by the addition of one or more terminal sialicacid-PEG moieties. The AT III molecule is first contacted with sialidaseto remove terminal sialic acid moieties. Then, the molecule is contactedwith a sialyltransferase and an appropriate sialic acid donor that hasbeen derivatized with a PEG moiety. In FIG. 54C, AT III expressed invarious mammalian systems is remodeled by the addition of sialicacid-PEG moieties. The AT III molecule is contacted with sialidase toremove terminal sialic acid moieties. The molecule is then contactedwith a ST3Gal3 and an appropriate sialic acid donor that has beenderivatized with a PEG moiety at 1.2 mol eq. The molecule is thencontacted with a ST3Gal3 and an appropriate sialic acid donor to capremaining terminal galactose moieties. In FIG. 54D, AT III is expressedin NSO murine myeloma cells is remodeled to have complex glycanmolecules with terminal sialic acid-PEG moieties. The AT III molecule iscontacted with sialidase and α-galactosidase to remove terminal sialicacid and galactose moieties. The molecule is then contacted withgalactosyltransferase and an appropriated galactose donor. The moleculeis then contacted with a ST3Gal3 and an appropriate sialic acid donorthat has been derivatized with a PEG moiety. In FIG. 54E, AT IIIexpressed in various mammalian systems is remodeled to have nearlycomplete terminal sialic acid-PEG moieties. The AT III molecule iscontacted with sialidase to remove terminal sialic acid moieties. Themolecule is then contacted with a ST3Gal3 and an appropriate sialic aciddonor that has been derivatized with a PEG moiety at 16 mol eq. Themolecule is then contacted with ST3Gal3 and an appropriate sialic aciddonor to cap remaining terminal galactose moieties. In FIG. 54F, AT IIIexpressed in various mammalian systems is remodeled by the addition ofone or more terminal sialic acid PEG moieties. The AT III molecule iscontacted with ST3Gal3 and an appropriate sialic acid donor that hasbeen derivatized with a levulinate moiety. The molecule is thencontacted with hydrazine-PEG. In FIG. 54G, AT III expressed in variousmammalian systems is remodeled by the addition of one or more terminalpoly-α2,8-linked sialic acid moieties. The AT III molecule is contactedwith poly-α2,8-sialyltransferase and an appropriate sialic acid donor.In FIG. 54I, AT III expressed in insect, yeast or fungi cells isremodeled by the addition of branching N—N-acetylglucosamine-PEGmoieties. The AT III molecule is contacted with GnT-I and an appropriateN-acetylglucosamine donor that has been derivatized with PEG. In FIG.54J, AT III expressed in yeast is remodeled by removing high mannoseglycan structures and the addition of terminal sialic acid-PEG moieties.The AT III molecule is contacted with endoglycanase to trim backglycosyl groups. The molecule is then contacted withgalactosyltransferase and an appropriate galactose donor. The moleculeis then contacted with ST3Gal3 and an appropriate sialic acid donor thathas been derivatized with a PEG moiety. In FIG. 54K, AT III expressed invarious mammalian systems is remodeled by the addition ofglycoconjugated transferrin. The AT III molecule is contacted withST3Gal3 and an appropriate sialic acid donor that has been derivatizedwith a linker-galactose donor moiety. The molecule is then contactedwith galactosyltransferase and endoglycanase-treated transferrin. InFIG. 54M, AT III expressed in yeast is remodeled by the removal ofmannose glyean structures and the addition of terminal galactose-PEGmoieties. The molecule is contacted with endoglycanase to trim backglycosyl groups. The molecule is further contacted withgalactosyltransferase and an appropriate galactose donor that has beenderivatized with a PEG moiety. In FIG. 54N, AT III expressed in plantcells is remodeled by converting the glycan structures intomammalian-type complex glycans and then adding one or more terminalgalactose-PEG moieties. The AT III molecule is contacted with xylosidaseto remove xylose residues. The molecule is then contacted withgalactosyltransferase and an appropriate galactose donor that has beenderivatized with a PEG moiety. In FIG. 54O, AT III expressed in variousmammalian systems is remodeled by the addition of one or more terminalsialic acid-PEG moieties to terminal galactose moieties. The AT IIImolecule is contacted with ST3Gal3 and an appropriate sialic acid PEGdonor that has been derivatized with PEG.

In another exemplary embodiment, the invention provides methods formodifying the α and β subunits of human Chorionic Gonadotropin (hCG).FIGS. 55A to 55J present some examples. In FIG. 55B, hCG expressed invarious mammalian and insect systems is remodeled by the addition ofterminal sialic acid-PEG moieties. The hCG molecule is contacted withsialidase to remove terminal sialic acid moieties. The molecule is thencontacted with ST3Gal3 and an appropriate sialic acid donor moleculethat has been derivatized with a PEG moiety. In FIG. 55C, hCG expressedin insect cell, yeast or fungi systems is remodeled by building out theN-linked glycans and the addition of terminal sialic acid-PEG moieties.The hCG molecule is contacted with GnT-I and GnT-II, and an appropriatedN-acetylglucosamine donor. The molecule is then contacted withgalactosyltransferase and an appropriate galactose donor. The moleculeis further contacted with ST3Gal3 and an appropriate sialic acid donorthat has been derivatized with a PEG moiety. In FIG. 55D, hCG expressedin various mammalian and insect systems is remodeled by the addition ofone or more terminal sialic acid-PEG moieties on O-linked glycanstructures. The hCG molecule is contacted with sialidase to removeterminal sialic acid moieties. The molecule is then contacted withST3Gal3 and an appropriate sialic acid donor to cap the glycanstructures with sialic acid moieties. The molecule is then contactedwith ST3Gal1 and an appropriate sialic acid donor that has beenderivatized with PEG. In FIG. 55E, hCG expressed in various mammalianand insect systems is remodeled by the addition of sialic acid-PEGmoieties to N-linked glycan structures. The hCG molecule is contactedwith ST3Gal3 and an appropriate sialic acid donor that has beenderivatized with PEG. In FIG. 55F, hCG expressed in insect cells, yeastor fungi, is remodeled by the addition of terminalN-acetylglucosamine-PEG molecules. The hCG molecule is contacted withGnT-I and GnT-II, and an appropriate N-acetylglucosamine donor that hasbeen derivatized with PEG. In FIG. 55G, hCG expressed in insect cells,yeast or fungi, is remodeled by the addition of not more than oneN-acetylglucosamine-PEG moiety per N-linked glycan structure. The hCGmolecule is contacted with GnT-I and an appropriate N-acetylglucosaminedonor that has been derivatized with a PEG moiety. In FIG. 55H, hCGexpressed in various mammalian systems is remodeled by the addition ofone or more terminal sialic acid-PEG moiety to O-linked glycanstructures. The hCG molecule is contacted with ST3Gal3 and anappropriate sialic acid donor that has been derivatized with PEG. InFIG. 55I, hCG expressed in various mammalian systems is remodeled by theaddition of terminal sialic acid-PEG moieties. The hCG molecule iscontacted with α2,8-SA and an appropriate sialic acid donor that hasbeen derivatized with a PEG moiety. In FIG. 55J, hCG expressed invarious mammalian systems is remodeled by the addition of terminalsialic acid moieties. The hCG molecule is contacted withpoly-alpha2,8-ST and an appropriate sialic acid donor that has beenderivatized with a PEG moiety.

In another exemplary embodiment, the invention provides methods formodifying alpha-galactosidase A (Fabrazyme™). FIGS. 56A to 56J presentsome examples. In FIG. 56B, alpha-galactosidase A expressed in andsecreted from various mammalian and insect systems is remodeled by theaddition of one or more terminal galactose-PEG-transferrin moieties. Thealpha-galactosidase A molecule is contacted with Endo-H to trim backglycosyl groups. The molecule is then contacted withgalactosyltransferase and an appropriate galactose donor that has beenderivatized with PEG and transferrin. In FIG. 56C, alpha-galactosidase Aexpressed in and secreted from various mammal and insect cell systems isremodeled by the addition of one or more terminal sialicacid-linker-mannose-6-phosphate moieties. The alpha-galactosidase Amolecule is contacted with sialidase to remove terminal sialic acidmoieties. The molecule is further contacted with ST3Gal3 and anappropriate sialic acid donor that has been conjugated via a linker tomannose-6-phosphate. In FIG. 56D, alpha-galactosidase A expressed in NSOmurine myeloma cells is remodeled by the addition of terminal sialicacid-linker-mannose-6-phosphate moieties. The alpha-galactosidase Amolecule is contacted with sialidase and α-galactosidase to removeterminal sialic acid and galactose moieties. The molecule is thencontacted with galactosyltransferase and an appropriate galactose donor.The molecule is then contacted with sialyltransferase and an appropriatesialic acid donor that has been conjugated via a linker tomannose-6-phosphate. In FIG. 56E, alpha-galactosidase A expressed in andsecreted from various mammalian and insect cell systems is remodeled bythe addition of one or more terminal sialic acid-PEG moieties. Thealpha-galactosidase A molecule is contacted with sialidase to removeterminal sialic acid moieties. The molecule is then contacted withsialyltransferase and an appropriate sialic acid donor that has beenderivatized with a PEG moiety. In FIG. 56F, alpha-galactosidase Aexpressed in mammalian, insect, yeast or fungi systems, is remodeled bythe addition of one or more terminal mannose-linker-ApoE moieties. Thealpha-galactosidase A molecule is contacted with mannosyltransferase andan appropriate mannose donor that has been conjugated via a linker toApoE. In FIG. 56G, alpha-galactosidase A expressed in mammalian, insect,yeast or fungal systems is remodeled by the addition ofgalactose-linker-alpha2-macroglobulin moieties. The alpha-galactosidaseA molecule is contacted with Endo-H to trim back glycosyl groups. Themolecule is then contacted with galactosyltransferase and an appropriategalactose donor that has been conjugated via a linker toalpha2-macroglobulin. In FIG. 56H, alpha-galactosidase A expressed ininsect, yeast and fungal systems, is remodeled by the addition of one ormore N-acetylglucosamine-PEG-mannose-6-phosphate moieties. Thealpha-galactosidase molecule is contacted with GnT-I and, an appropriateN-acetyl-glucosamine donor that has been derivatized with PEG andmannose-6-phosphate. In FIG. 561, alpha-galactosidase A expressed ininsect, yeast or fungal systems, is remodeled by the addition of one ormore terminal galactose-PEG-transferrin moieties. Thealpha-galactosidase A molecule is contacted with GnT-I and anappropriate N-acetyl-glucosamine donor. The molecule is then contactedwith galactosyltransferase and an appropriate galactose donor that hasbeen derivatized with PEG and transferrin. In FIG. 56J,alpha-galactosidase A expressed in insect, yeast or fungi systems isremodeled by the addition of one or more terminal sialicacid-PEG-melanotransferrin moieties. The alpha-galactosidase A moleculeis contacted with GnT-I and GnT-II and an appropriateN-acetyl-glucosamine donor. The molecule is then contacted withgalactosyltransferase and an appropriate galactose donor. The moleculeis then contacted with sialyltransferase and an appropriate sialic aciddonor that has been derivatized with PEG and melanotransferrin.

In another exemplary embodiment, the invention provides methods formodifying alpha-iduronidase (Aldurazyme™). FIGS. 57A to 57J present someexamples. In FIG. 57B, alpha-iduronidase expressed in and secreted fromvarious mammalian and insect systems is remodeled by the addition of oneor more terminal galactose-PEG-transferrin moieties. Thealpha-iduronidase molecule is contacted with Endo-H to trim backglycosyl groups. The molecule is then contacted withgalactosyltransferase and an appropriate galactose donor that has beenderivatized with PEG and transferrin. In FIG. 57C, alpha-iduronidaseexpressed in and secreted from various mammal and insect cell systems isremodeled by the addition of terminal sialicacid-linker-mannose-6-phosphate moieties. The alpha-iduronidase moleculeis contacted with sialidase to remove terminal sialic acid moieties. Themolecule is then contacted with ST3Gal3 and an appropriate sialic aciddonor that has been conjugated via a linker to mannose-6-phosphate. InFIG. 57D, alpha-iduronidase expressed in NSO murine myeloma cells isremodeled by the addition of one or more terminal sialicacid-linker-mannose-6-phosphate moieties. The alpha-iduronidase moleculeis contacted with sialidase and α-galactosidase to remove terminalsialic acid and galactose moieties. The molecule is then contacted withgalactosyltransferase and an appropriate galactose donor. The moleculeis further contacted with sialyltransferase and an appropriate sialicacid donor that has been conjugated via a linker to mannose-6-phosphate.In FIG. 57E, alpha-iduronidase expressed in and secreted from variousmammalian and insect cell systems is remodeled by the addition of one ormore terminal sialic acid-PEG moieties. The alpha-iduronidase moleculeis contacted with sialidase to remove terminal sialic acid moieties. Themolecule is further contacted with sialyltransferase and an appropriatesialic acid donor that has been derivatized with a PEG moiety. In FIG.57F, alpha-iduronidase expressed in mammalian, insect, yeast or fungisystems is remodeled by the addition of one or more terminalmannose-linker-ApoE moieties. The alpha-iduronidase molecule iscontacted with mannosyltransferase and an appropriate mannose donor thathas been conjugated via a linker to ApoE. In FIG. 57G, alpha-iduronidaseexpressed in mammalian, insect, yeast or fungal systems is remodeled bythe addition of one or more galactose-linker-alpha2-macroglobulinmoieties. The alpha-iduronidase molecule is contacted with Endo-H totrim back glycosyl groups. The molecule is then contacted withgalactosyltransferase and an appropriate galactose donor that has beenconjugated via a linker to alpha2-macroglobulin. In FIG. 57H,alpha-iduronidase expressed in insect, yeast and fungal systems, isremodeled by the addition of one or moreN-acetylglucosamine-PEG-mannose-6-phosphate moieties. Thealpha-galactosidase molecule is contacted with GnT-I and an appropriateN-acetyl-glucosamine donor that has been derivatized with PEG andmannose-6-phosphate. In FIG. 57I, alpha-iduronidase expressed in insect,yeast or fungal systems, is remodeled by the addition of one or moreterminal galactose-PEG-transferrin moieties. The alpha-iduronidasemolecule is contacted with GnT-I and an appropriate N-acetyl-glucosaminedonor. The molecule is then contacted with galactosyltransferase and anappropriate galactose donor that has been derivatized with PEG andtransferrin. In FIG. 57J, alpha-iduronidase expressed in insect, yeastor fungi systems, is remodeled by the addition of one or more terminalsialic acid-PEG-melanotransferrin moieties. The alpha-iduronidasemolecule is contacted with GnT-I and GnT-II and an appropriateN-acetyl-glucosamine donor. The molecule is then contacted withgalactosyltransferase and an appropriate galactose donor. The moleculeis further contacted with sialyltransferase and an appropriate sialicacid donor that has been derivatized with PEG and melanotransferrin.

A. Creation or Elimination of N-Linked Glycosylation Sites

The present invention contemplates the use of peptides in which the siteof the glycan chain(s) on the peptide have been altered from that of thenative peptide. Typically, N-linked glycan chains are linked to theprimary peptide structure at asparagine residues where the asparagineresidue is within an amino acid sequence that is recognized by amembrane-bound glycosyltransferase in the endoplasmic reticulum (ER).Typically, the recognition site on the primary peptide structure is thesequence asparagine-X-serine/threonine where X can be any amino acidexcept proline and aspartic acid. While this recognition site istypical, the invention further encompasses peptides that have N-linkedglycan chains at other recognition sites where the N-linked chains areadded using natural or recombinant glycosyltransferases.

Since the recognition site for N-linked glycosylation of a peptide isknown, it is within the skill of persons in the art to create mutatedprimary peptide sequences wherein a native N-linked glycosylationrecognition site is removed, or alternatively or in addition, one ormore additional N-glycosylation recognition sites are created. Mostsimply, an asparagine residue can be removed from the primary sequenceof the peptide thereby removing the attachment site for a glycan, thusremoving one glycan from the mature peptide. For example, a nativerecognition site with the sequence of asparagie-serine-serine can begenetically engineered to have the sequence leucine-serine-serine, thuseliminating a N-linked glycosylation site at this position.

Further, an N-linked glycosylation site can be removed by altering theresidues in the recognition site so that even though the asparagineresidue is present, one or more of the additional recognition residuesare absent. For example, a native sequence of asparagine-serine-serinecan be mutated to asparagine-serine-lysine, thus eliminating anN-glycosylation site at that position. In the case of N-linkedglycosylation sites comprising residues other than the typicalrecognition sites described above, the skilled artisan can determine thesequence and residues required for recognition by the appropriateglycosyltransferase, and then mutate at least one residue so theappropriate glycosyltransferase no longer recognizes that site. In otherwords, it is well within the skill of the artisan to manipulate theprimary sequence of a peptide such that glycosylation sites are eithercreated or are removed, or both, thereby generating a peptide having analtered glycosylation pattern. The invention should therefore not beconstrued to be limited to any primary peptide sequence provided hereinas the sole sequence for glycan remodeling, but rather should beconstrued to include any and all peptide sequences suitable for glycanremodeling.

To create a mutant peptide, the nucleic acid sequence encoding theprimary sequence of the peptide is altered so that native codonsencoding native amino acid residues are mutated to generate a codonencoding another amino acid residue. Techniques for altering nucleicacid sequence are common in the art and are described for example in anywell-known molecular biology manual.

In addition, the nucleic acid encoding a primary peptide structure canbe synthesized in vitro, using standard techniques. For example, anucleic acid molecule can be synthesized in a “gene machine” usingprotocols such as the phosphoramidite method. If chemically-synthesizeddouble stranded DNA is required for an application such as the synthesisof a nucleic acid or a fragment thereof, then each complementary strandis synthesized separately. The production of short nucleic acids (60 to80 base pairs) is technically straightforward and can be accomplished bysynthesizing the complementary strands and then annealing them. For theproduction of longer nucleic acids (>300 base pairs), special strategiesmay be required, because the coupling efficiency of each cycle duringchemical DNA synthesis is seldom 100%. To overcome this problem,synthetic genes (double-stranded) are assembled in modular form fromsingle-stranded fragments that are from 20 to 100 nucleotides in length.For reviews on polynucleotide synthesis, see, for example, Glick andPasternak (Molecular Biotechnology, Principles and Applications ofRecombinant DNA, 1994, ASM Press), Itakura et al. (1984, Annu. Rev.Biochem. 53:323), and Climie et al. (1990, Proc. Nat'l Acad. Sci. USA87:633).

Additionally, changes in the nucleic acid sequence encoding the peptidecan be made by site-directed mutagenesis. As will be appreciated, thistechnique typically employs a phage vector which exists in both a singlestranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily available and their use is generally well known tothose skilled in the art. Double stranded plasmids are also routinelyemployed in site-directed mutagenesis which eliminates the step oftransferring the nucleic acid of interest from a plasmid to a phage.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector or melting the two strands of a double strandedvector which includes within its sequence a DNA sequence which encodesthe desired peptide. An oligonucleotide primer bearing the desiredmutated sequence is prepared generally synthetically. This primer isthen annealed with the single-stranded vector, and subjected to DNApolymerizing enzymes such as E. coli polymerase I Klenow fragment, inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform or transfectappropriate cells, such as E. coli cells, and clones are selected whichinclude recombinant vectors bearing the mutated sequence arrangement. Agenetic selection scheme was devised by Kunkel et al. (1987, Kunkel etal., Methods Enzymol. 154:367-382) to enrich for clones incorporatingthe mutagenic oligonucleotide. Alternatively, the use of PCR™ withcommercially available thermostat enzymes such as Taq polymerase may beused to incorporate a mutagenic oligonucleotide primer into an amplifiedDNA fragment that can then be cloned into an appropriate cloning orexpression vector. The PCR™-mediated mutagenesis procedures of Tomic etal. (1990, Nucl. Acids Res., 12:1656) and Upender et al. (1995,Biotechniques, 18:29-31) provide two examples of such protocols. A PCR™employing a thermostable ligase in addition to a thermostable polymerasemay also be used to incorporate a phosphorylated mutagenicoligonucleotide into an amplified DNA fragment that may then be clonedinto an appropriate cloning or expression vector. The mutagenesisprocedure described by Michael (1994, Biotechniques 16:410-412) providesan example of one such protocol.

Not all Asn-X-Ser/Thr sequences are N-glycosylated suggesting thecontext in which the motif is presented is important. In anotherapproach, libraries of mutant peptides having novel N-linked consensussites are created in order to identify novel N-linked sites that areglycosylated in vivo and are beneficial to the activity, stability orother characteristics of the peptide.

As noted previously, the consensus sequence for the addition of N-Linkedglycan chains in glycoproteins is Asn-X-Ser/Thr where X can be any aminoacid. The nucleotide sequence encoding the amino acid two positions tothe carboxyl terminal side of the Asn may be mutated to encode a Serand/or Thr residue using standard procedures known to those of ordinaryskill in the art. As stated above not all Asn-X-Ser/Thr sites aremodified by the addition of glycans. Therefore, each recombinant mutatedglycoprotein must be expressed in a fungal, yeast or animal or mammalianexpression system and analyzed for the addition of an N-linked glycanchain. The techniques for the characterization of glycosylation sitesare well known to one skilled in the art. Further, the biologicalfunction of the mutated recombinant glycoprotein can be determined usingassays standard for the particular protein being examined. Thus, itbecomes a simple matter to manipulate the primary sequence of a peptideand identify novel glycosylation sites contained therein, and furtherdetermine the effect of the novel site on the biological activity of thepeptide.

In an alternative embodiment, the nucleotide sequence encoding the aminoacid two positions to the amino terminal side of Ser/Thr residues may bemutated to encode an Asn using standard procedures known to those ofordinary skill in the art. The procedures to determine whether a novelglycosylation site has been created and the effect of this site on thebiological activity of the peptide are described above.

B. Creation or Elimination of O-Linked Glycosylation Sites

The addition of an O-linked glycosylation site to a peptide isconveniently accomplished by altering the primary amino acid sequence ofthe peptide such that it contains one or more additional O-linkedglycosylation sites compared with the beginning primary amino acidsequence of the peptide. The addition of an O-linked glycosylation siteto the peptide may also be accomplished by incorporation of one or moreamino acid species into the peptide which comprises an —OH group,preferably serine or threonine residues, within the sequence of thepeptide, such that the OH group is accessible and available for O-linkedglycosylation. Similar to the discussion of alteration of N-linkedglycosylation sites in a peptide, the primary amino acid sequence of thepeptide is preferably altered at the nucleotide level. Specificnucleotides in the DNA sequence encoding the peptide may be altered suchthat a desired amino acid is encoded by the sequence. Mutation(s) in DNAare preferably made using methods known in the art, such as thetechniques of phosphoramidite method DNA synthesis and site-directedmutagenesis described above.

Alternatively, the nucleotide sequence encoding a putative site forO-linked glycan addition can be added to the DNA molecule in one orseveral copies to either 5′ or the 3′ end of the molecule. The alteredDNA sequence is then expressed in any one of a fungal, yeast, or animalor mammalian expression system and analyzed for the addition of thesequence to the peptide and whether or not this sequence is a functionalO-linked glycosylation site. Briefly, a synthetic peptide acceptorsequence is introduced at either the 5′ or 3′ end of the nucleotidemolecule. In principle, the addition of this type of sequence is lessdisruptive to the resulting glycoprotein when expressed in a suitableexpression system. The altered DNA is then expressed in CHO cells orother suitable expression system and the proteins expressed thereby areexamined for the presence of an O-linked glycosylation site. Inaddition, the presence or absence of glycan chains can be determined.

In yet another approach, advantageous sites for new O-linked sites maybe found in a peptide by creating libraries of the peptide containingvarious new O-linked sites. For example, the consensus amino acidsequence for N-acetylgalactosamine addition by anN-acetylgalactosaminyltransferase depends on the specific transferaseused. The amino acid sequence of a peptide may be scanned to identifycontiguous groups of amino acids that can be mutated to generatepotential sites for addition of O-linked glycan chains. These mutationscan be generated using standard procedures known to those of ordinaryskill in the art as described previously. In order to determine if anydiscovered glycosylation site is actually glycosylated, each recombinantmutated peptide is then expressed in a suitable expression system and issubsequently analyzed for the addition of the site and/or the presenceof an O-linked glycan chain.

C. Chemical Synthesis of Peptides

While the primary structure of peptides useful in the invention can begenerated most efficiently in a cell-based expression system, it iswithin the scope of the present invention that the peptides may begenerated synthetically. Chemical synthesis of peptides is well known inthe art and include, without limitation, stepwise solid phase synthesis,and fragment condensation either in solution or on solid phase. Aclassic stepwise solid phase synthesis of involves covalently linking anamino acid corresponding to the carboxy-terminal amino acid of thedesired peptide chain to a solid support and extending the peptide chaintoward the amino end by stepwise coupling of activated amino acidderivatives having activated carboxyl groups. After completion of theassembly of the fully protected solid phase bound peptide chain, thepeptide-solid phase covalent attachment is cleaved by suitable chemistryand the protecting groups are removed to yield the product peptide. See,R. Merrifield, Solid Phase Peptide Synthesis: The Synthesis of aTetrapeptide, J. Am. Chem. Soc., 85:2149-2154 (1963). The longer thepeptide chain, the more challenging it is to obtain high-puritywell-defined products. Due to the production of complex mixtures, thestepwise solid phase synthesis approach has size limitations. Ingeneral, well-defined peptides of 100 contiguous amino acid residues ormore are not routinely prepared via stepwise solid phase synthesis.

The segment condensation method involves preparation of several peptidesegments by the solid phase stepwise method, followed by cleavage fromthe solid phase and purification of these maximally protected segments.The protected segments are condensed one-by-one to the first segment,which is bound to the solid phase.

The peptides useful in the present invention may be synthesized byexclusive solid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. These synthesis methodsare well-known to those of skill in the art (see, for example,Merrifield, J. Am. Chem. Soc. 85:2149 (1963), Stewart et al., “SolidPhase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co. 1984),Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986), Atherton et al., SolidPhase Peptide Synthesis: A Practical Approach (IRL Press 1989), Fieldsand Colowick, “Solid-Phase Peptide Synthesis,” Methods in EnzymologyVolume 289 (Academic Press 1997), and Lloyd-Williams et al., ChemicalApproaches to the Synthesis of Peptides and Peptides (CRC Press, Inc.1997)). Variations in total chemical synthesis strategies, such as“native chemical ligation” and “expressed peptide ligation” are alsostandard (see, for example, Dawson et al., Science 266:776 (1994),Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997), Dawson,Methods Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l Acad. Sci. USA95:6705 (1998), and Severinov and Muir, J. Biol. Chem. 273:16205(1998)). Also useful are the solid phase peptide synthesis methodsdeveloped by Gryphon Sciences, South San Francisco, Calif. See, U.S.Pat. Nos. 6,326,468, 6,217,873, 6,174,530, and 6,001,364, all of whichare incorporated in their entirety by reference herein.

D. Post-Translational Modifications

It will be appreciated to one of ordinary skill in the art that peptidesmay undergo post-translational modification besides the addition ofN-linked and/or O-linked glycans thereto. It is contemplated thatpeptides having post-translational modifications other thanglycosylation can be used as peptides in the invention, as long as thedesired biological activity or function of the peptide is maintained orimproved. Such post-translational modifications may be naturalmodifications usually carried out in vivo, or engineered modificationsof the peptide carried out in vitro. Contemplated known modificationsinclude, but are not limited to, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent crosslinks, formation of cysteine, formation of pyroglutamate,formylation, gamma carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto peptides such as arginylation, and ubiquitination. Enzymes that maybe used to carry out many of these modifications are well known in theart, and available commercially from companies such as BoehringerMannheim (Indianapolis, Ind.) and Sigma Chemical Company (St. Louis,Mo.), among others.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Peptides—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Post-translational Covalent Modification of Peptides, B. C. Johnson,Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth.Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci.663:48-62 (1992)).

Covalent modifications of a peptide may also be introduced into themolecule in vitro by reacting targeted amino-acid residues of thepeptide with an organic derivatizing agent that is capable of reactingwith selected side chains or terminal amino-acid residues. Most commonlyderivatized residues are cysteinyl, histidyl, lysinyl, arginyl, tyrosyl,glutaminyl, asparaginyl and amino terminal residues. Hydroxylation ofproline and lysine, phosphorylation of hydroxyl groups of seryl andthreonyl residues, methylation of the alpha-amino groups of lysine,histidine, and histidine side chains, acetylation of the N-terminalamine and amidation of the C-terminal carboxylic groups. Suchderivatized moieties may improve the solubility, absorption, biologicalhalf life and the like. The moieties may also eliminate or attenuate anyundesirable side effect of the peptide and the like.

In addition, derivatization with bifunctional agents is useful forcross-linking the peptide to water insoluble support matrices or toother macromolecular carriers. Commonly used cross-linking agentsinclude glutaraldehyde, N-hydroxysuccinimide esters, homobifunctionalimidoesters, 1,1-bis(-diazoloacetyl)-2-phenylethane, and bifunctionalmaleimides. Derivatizing agents such asmethyl-3-[9p-azidophenyl)]dithiopropioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287 and 3,691,016 may be employed for peptideimmobilization.

E. Fusion Peptides/Peptides

Peptides useful in the present invention may comprise fusion peptides.Fusion peptides are particularly advantageous where biological and/orfunctional characteristics of two peptides are desired to be combined inone peptide molecule. Such fusion peptides can present combinations ofbiological activity and function that are not found in nature to createnovel and useful molecules of therapeutic and industrial applications.Biological activities of interest include, but are not limited to,enzymatic activity, receptor and/or ligand activity, immunogenic motifs,and structural domains.

Such fusion peptides are well known in the art, and the methods ofcreation will be well-known to those in the art. For example, a humanα-interferon—human albumin fusion peptide has been made wherein theresulting peptide has the therapeutic benefits of α-interferon combinedwith the long circulating life of albumin, thereby creating atherapeutic composition that allows reduced dosing frequency andpotentially reduced side effects in patients. See, Albuferon™ from HumanGenome Sciences, Inc. and U.S. Pat. No. 5,766,883. Other fusion peptidesinclude antibody molecules that are described elsewhere herein.

F. Generation of Smaller “Biologically Active” Molecules

The peptides used in the invention may be variants of native peptides,wherein a fragment of the native peptide is used in place of the fulllength native peptide. In addition, pre-pro-, and pre-peptides arecontemplated. Variant peptides may be smaller in size that the nativepeptide, and may comprise one or more domains of a larger peptide.Selection of specific peptide domains can be advantageous when thebiological activity of certain domains in the peptide is desired, butthe biological activity of other domains in the peptide is not desired.Also included are truncations of the peptide and internal deletionswhich may enhance the desired therapeutic effect of the peptide. Anysuch forms of a peptide is contemplated to be useful in the presentinvention provided that the desired biological activity of the peptideis preserved.

Shorter versions of peptides may have unique advantages not found in thenative peptide. In the case of human albumin, it has been found that atruncated form comprising as little as 63% of the native albumin peptideis advantageous as a plasma volume expander. The truncated albuminpeptide is considered to be better than the native peptide for thistherapeutic purpose because an individual peptide dose of only one-halfto two-thirds that of natural-human serum albumin, or recombinant humanserum albumin is required for the equivalent colloid osmotic effect. SeeU.S. Pat. No. 5,380,712, the entirety of which is incorporated byreference herein.

Smaller “biologically active” peptides have also been found to haveenhanced therapeutic activity as compared to the native peptide. Thetherapeutic potential of IL-2 is limited by various side effectsdominated by the vascular leak syndrome. A shorter chemicallysynthesized version of the peptide consisting of residues 1-30corresponding to the entire α-helix was found to fold properly andcontain the natural IL-2 biological activity with out the attending sideeffects.

G. Generation of Novel Peptides

The peptide of the invention may be derived from a primary sequence of anative peptide, or may be engineered using any of the many means knownto those of skill in the art. Such engineered peptides can be designedand/or selected because of enhanced or novel properties as compared withthe native peptide. For example, peptides may be engineered to haveincreased enzyme reaction rates, increased or decreased binding affinityto a substrate or ligand, increased or decreased binding affinity to areceptor, altered specificity for a substrate, ligand, receptor or otherbinding partner, increased or decreased stability in vitro and/or invivo, or increased or decreased immunogenicity in an animal.

H. Mutations

1. Rational Design Mutation

The peptides useful in the methods of the invention may be mutated toenhance a desired biological activity or function, to diminish anundesirable property of the peptide, and/or to add novel activities orfunctions to the peptide. “Rational peptide design” may be used togenerate such altered peptides. Once the amino acid sequence andstructure of the peptide is known and a desired mutation planned, themutations can be made most conveniently to the corresponding nucleicacid codon which encodes the amino acid residue that is desired to bemutated. One of skill in the art can easily determine how the nucleicacid sequence should be altered based on the universal genetic code, andknowledge of codon preferences in the expression system of choice. Amutation in a codon may be made to change the amino acid residue thatwill be polymerized into the peptide during translation. Alternatively,a codon may be mutated so that the corresponding encoded amino acidresidue is the same, but the codon choice is better suited to thedesired peptide expression system. For example, cys-residues may bereplaced with other amino acids to remove disulfide bonds from themature peptide, catalytic domains may be mutated to alter biologicalactivity, and in general, isoforms of the peptide can be engineered.Such mutations can be point mutations, deletions, insertions andtruncations, among others.

Techniques to mutate specific amino acids in a peptide are well known inthe art. The technique of site-directed mutagenesis, discussed above, iswell suited for the directed mutation of codons. Theoligonucleotide-mediated mutagenesis method is also discussed in detailin Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York, starting at page 15.51). Systematicdeletions, insertions and truncations can be made using linker insertionmutagenesis, digestion with nuclease Bal31, and linker-scanningmutagenesis, among other method well known to those in the art (Sambrooket al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York).

Rational peptide design has been successfully used to increase thestability of enzymes with respect to thermoinactivation and oxidation.For example, the stability of an enzyme was improved by removal ofasparagine residues in a-amylase (Declerek et al., 2000, J. Mol. Biol.301:1041-1057), the introduction of more rigid structural elements suchas proline into α-amylase (Igarashi et al., 1999, Biosci. Biotechnol.Biochem. 63:1535-1540) and D-xylose isomerase (Zhu et al., 1999, PeptideEng. 12:635-638). Further, the introduction of additional hydrophobiccontacts stabilized 3-isopropylmalate dehydrogenase (Akanuma et al.,1999, Eur. J. Biochem. 260:499-504) and formate dehydrogenase obtainedfrom Pseudomonas sp. (Rojkova et al., 1999, FEBS Lett. 445:183-188). Themechanisms behind the stabilizing effect of these mutations is generallyapplicable to many peptides. These and similar mutations arecontemplated to be useful with respect to the peptides remodeled in themethods of the present invention.

2. Random Mutagenesis Techniques

Novel peptides useful in the methods of the invention may be generatedusing techniques that introduce random mutations in the coding sequenceof the nucleic acid. The nucleic acid is then expressed in a desiredexpression system, and the resulting peptide is assessed for propertiesof interest. Techniques to introduce random mutations into DNA sequencesare well known in the art, and include PCR mutagenesis, saturationmutagenesis, and degenerate oligonucleotide approaches. See Sambrook andRussell (2001, Molecular Cloning, A Laboratory Approach, Cold SpringHarbor Press, Cold Spring Harbor, NY) and Ausubel et al. (2002, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY).

In PCR mutagenesis, reduced Taq polymerase fidelity is used to introducerandom mutations into a cloned fragment of DNA (Leung et al., 1989,Technique 1:11-15). This is a very powerful and relatively rapid methodof introducing random mutations into a DNA sequence. The DNA region tobe mutagenized is amplified using the polymerase chain reaction (PCR)under conditions that reduce the fidelity of DNA synthesis by Taq DNApolymerase, e.g., by using an altered dGTP/dATP ratio and by adding Mn²⁺to the PCR reaction. The pool of amplified DNA fragments are insertedinto appropriate cloning vectors to provide random mutant libraries.

Saturation mutagenesis allows for the rapid introduction of a largenumber of single base substitutions into cloned DNA fragments (Mayers etal., 1985, Science 229:242). This technique includes generation ofmutations, e.g., by chemical treatment or irradiation of single-strandedDNA in vitro, and synthesis of a complementary DNA strand. The mutationfrequency can be modulated by modulating the severity of the treatment,and essentially all possible base substitutions can be obtained. Becausethis procedure does not involve a genetic selection for mutantfragments, both neutral substitutions as well as those that alterfunction, are obtained. The distribution of point mutations is notbiased toward conserved sequence elements.

A library of nucleic acid homologs can also be generated from a set ofdegenerate oligonucleotide sequences. Chemical synthesis of a degenerateoligonucleotide sequences can be carried out in an automatic DNAsynthesizer, and the synthetic genes may then be ligated into anappropriate expression vector. The synthesis of degenerateoligonucleotides is known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rdCleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevierpp. 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakuraet al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res.11:477. Such techniques have been employed in the directed evolution ofother peptides (see, for example, Scott et al. (1990) Science249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al.(1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; aswell as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

a. Directed Evolution

Peptides useful in the methods of the invention may also be generatedusing “directed evolution” techniques. In contrast to site directedmutagenesis techniques where knowledge of the structure of the peptideis required, there now exist strategies to generate libraries ofmutations from which to obtain peptides with improved properties withoutknowledge of the structural features of the peptide. These strategiesare generally known as “directed evolution” technologies and aredifferent from traditional random mutagenesis procedures in that theyinvolve subjecting the nucleic acid sequence encoding the peptide ofinterest to recursive rounds of mutation, screening and amplification.

In some “directed evolution” techniques, the diversity in the nucleicacids obtained is generated by mutation methods that randomly createpoint mutations in the nucleic acid sequence. The point mutationtechniques include, but are not limited to, “error-prone PCR™” (Caldwelland Joyce, 1994; PCR Methods Appl. 2:28-33; and Ke and Madison, 1997,Nucleic Acids Res. 25: 3371-3372), repeated oligonucleotide-directedmutagenesis (Reidhaar-Olson et al., 1991, Methods Enzymol. 208:564-586),and any of the aforementioned methods of random mutagenesis.

Another method of creating diversity upon which directed evolution canact is the use of mutator genes. The nucleic acid of interest iscultured in a mutator cell strain the genome of which typically encodesdefective DNA repair genes (U.S. Pat. No. 6,365,410; Selifonova et al.,2001, Appl. Environ. Microbiol. 67:3645-3649; Long-McGie et al., 2000,Biotech. Bioeng. 68:121-125; see, Genencor International Inc, Palo AltoCalif.).

Achieving diversity using directed evolution techniques may also beaccomplished using saturation mutagenesis along with degenerate primers(Gene Site Saturation Mutagenesis™, Diversa Corp., San Diego, Calif.).In this type of saturation mutagenesis, degenerate primers designed tocover the length of the nucleic acid sequence to be diversified are usedto prime the polymerase in PCR reactions. In this manner, each codon ofa coding sequence for an amino acid may be mutated to encode each of theremaining common nineteen amino acids. This technique may also be usedto introduce mutations, deletions and insertions to specific regions ofa nucleic acid coding sequence while leaving the rest of the nucleicacid molecule untouched. Procedures for the gene saturation techniqueare well known in the art, and can be found in U.S. Pat. No. 6,171,820.

b. DNA Shuffling

Novel peptides useful in the methods of the invention may also begenerated using the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”). DNA shuffling techniques are may be employed to modulatethe activities of peptides useful in the invention and may be used togenerate peptides having altered activity. See, generally, U.S. Pat.Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, andStemmer et al. (1994, Nature 370(6488):389-391); Crameri et al. (1998,Nature 391 (6664):288-291); Zhang et al. (1997, Proc. Natl. Acad. Sci.USA 94(9):4504-4509); Stemmer et al. (1994, Proc. Natl. Acad. Sci USA91(22):10747-10751), Patten et al. (1997, Curr. Opinion Biotechnol.8:724-33); Harayama, (1998, Trends Biotechnol. 16(2):76-82); Hansson, etal., (1999, J. Mol. Biol. 287:265-76); and Lorenzo and Blasco (1998,Biotechniques 24(2):308-13) (each of these patents are herebyincorporated by reference in its entirety).

DNA shuffling involves the assembly of two or more DNA segments byhomologous or site-specific recombination to generate variation in thepolynucleotide sequence. DNA shuffling has been used to generate novelvariations of human immunodeficiency virus type 1 proteins (Pekrun etal., 2002, J. Virol. 76(6):2924-35), triazine hydrolases (Raillard etal. 2001, Chem Biol 8(9):891-898), murine leukemia virus (MLV) proteins(Powell et al. 2000, Nat Biotechnol 18(12):1279-1282), andindoleglycerol phosphate synthase (Merz et al. 2000, Biochemistry39(5):880-889).

The technique of DNA shuffling was developed to generate biomoleculardiversity by mimicking natural recombination by allowing in vitrohomologous recombination of DNA (Stemmler, 1994, Nature 370: 389-391;and Stemmler, 1994, PNAS 91: 10747-10751). Generally, in this method apopulation of related genes is fragmented and subjected to recursivecycles of denaturation, rehybridization, followed by the extension ofthe 5′ overhangs by Taq polymerase. With each cycle, the length of thefragments increases, and DNA recombination occurs when fragmentsoriginating from different genes hybridize to each other. The initialfragmentation of the DNA is usually accomplished by nuclease digestion,typically using DNase (see Stemmler references, above), but may also beaccomplished by interrupted PCR synthesis (U.S. Pat. No. 5,965,408,incorporated herein by reference in its entirety; see, Diversa Corp.,San Diego, Calif.). DNA shuffling methods have advantages over randompoint mutation methods in that direct recombination of beneficialmutations generated by each round of shuffling is achieved and there istherefore a self selection for improved phenotypes of peptides.

The techniques of DNA shuffling are well known to those in art. Detailedexplanations of such technology is found in Stemmler, 1994, Nature 370:389-391 and Stemmler, 1994, PNAS 91: 10747-10751. The DNA shufflingtechnique is also described in U.S. Pat. Nos. 6,180,406, 6,165,793,6,132,970, 6,117,679, 6,096,548, 5,837,458, 5,834,252, 5,830,721,5,811,238, and 5,605,793 (all of which are incorporated by referenceherein in their entirety).

The art also provides even more recent modifications of the basictechnique of DNA shuffling. In one example, exon shuffling, exons orcombinations of exons that encode specific domains of peptides areamplified using chimeric oligonucleotides. The amplified molecules arethen recombined by self-priming PCR assembly (Kolkman and Stemmler,2001, Nat. Biotech. 19:423-428). In another example, using the techniqueof random chimeragenesis on transient templates (RACHITT) libraryconstruction, single stranded parental DNA fragments are annealed onto afull-length single-stranded template (Coco et al., 2001, Nat.Biotechnol. 19:354-359). In yet another example, staggered extensionprocess (StEP), thermocycling with very abbreviated annealing/extensioncycles is employed to repeatedly interrupt DNA polymerization fromflanking primers (Zhao et al., 1998, Nat. Biotechnol. 16: 258-261). Inthe technique known as CLERY, in vitro family shuffling is combined within vivo homologous recombination in yeast (Abecassis et al., 2000,Nucleic Acids Res. 28:E88; ). To maximize intergenic recombination,single stranded DNA from complementary strands of each of the nucleicacids are digested with DNase and annealed (Kikuchi et al., 2000, Gene243:133-137). The blunt ends of two truncated nucleic acids of variablelengths that are linked by a cleavable sequence are then ligated togenerate gene fusion without homologous recombination (Sieber et al.,2001, Nat Biotechnol. 19:456-460; Lutz et al., 2001, Nucleic Acids Res.29:E16; Ostermeier et al., 1999, Nat. Biotechnol. 17:1205-1209; Lutz andBenkovic, 2000, Curr. Opin. Biotechnol. 11:319-324). Recombinationbetween nucleic acids with little sequence homology in common has alsobeen enhanced using exonuclease-mediated blunt-ending of DNA fragmentsand ligating the fragments together to recombine them (U.S. Pat. No.6,361,974, incorporated herein by reference in its entirety). Theinvention contemplates the use of each and every variation describedabove as a means of enhancing the biological properties of any of thepeptides and/or enzymes useful in the methods of the invention.

In addition to published protocols detailing directed evolution and geneshuffling techniques, commercial services are now available that willundertake the gene shuffling and selection procedures on peptides ofchoice. Maxygen (Redwood City, Calif.) offers commercial services togenerate custom DNA shuffled libraries. In addition, this company willperform customized directed evolution procedures including geneshuffling and selection on a peptide family of choice.

Optigenix, Inc. (Newark, Del.) offers the related service of plasmidshuffling. Optigenix uses families of genes to obtain mutants thereinhaving new properties. The nucleic acid of interest is cloned into aplasmid in an Aspergillus expression system. The DNA of the relatedfamily is then introduced into the expression system and recombinationin conserved regions of the family occurs in the host. Resulting mutantDNAs are then expressed and the peptide produced therefrom are screenedfor the presence of desired properties and the absence of undesiredproperties.

c. Screening Procedures

Following each recursive round of “evolution,” the desired peptidesexpressed by mutated genes are screened for characteristics of interest.The “candidate” genes are then amplified and pooled for the next roundof DNA shuffling. The screening procedure used is highly dependant onthe peptide that is being “evolved” and the characteristic of interest.Characteristics such as peptide stability, biological activity,antigenicity, among others can be selected using procedures that arewell known in the art. Individual assays for the biological activity ofpreferred peptides useful in the methods of the invention are describedelsewhere herein.

d. Combinations of Techniques

It will be appreciated by the skilled artisan that the above techniquesof mutation and selection can be combined with each other and withadditional procedures to generate the best possible peptide moleculeuseful in the methods of the invention. Thus, the invention is notlimited to any one method for the generation of peptides, and should beconstrued to encompass any and all of the methodology described herein.For example, a procedure for introducing point mutations into a nucleicacid sequence may be performed initially, followed by recursive roundsof DNA shuffling, selection and amplification. The initial introductionof point mutations may be used to introduce diversity into a genepopulation where it is lacking, and the following round of DNA shufflingand screening will select and recombine advantageous point mutations.

III. Glycosidases and Glycotransferases

A. Glycosidases

Glycosidases are glycosyltransferases that use water as an acceptormolecule, and as such, are typically glycoside-hydrolytic enzymes.Glycosidases can be used for the formation of glycosidic bonds in vitroby controlling the thermodynamics or kinetics of the reaction mixture.Even with modified reaction conditions, though, glycosidase reactionscan be difficult to work with, and glycosidases tend to give lowsynthetic yields as a result of the reversible transglycosylase reactionand the competing hydrolytic reaction.

A glycosidase can function by retaining the stereochemistry at the bondbeing broken during hydrolysis or by inverting the stereochemistry atthe bond being broken during hydrolysis, classifying the glycosidase aseither a “retaining” glycosidase or an “inverting” glycosidase,respectively. Retaining glycosidases have two critical carboxylic acidmoieties present in the active site, with one carboxylate acting as anacid/base catalyst and the other as a nucleophile, whereas with theinverting glycosidases, one carboxylic acid functions as an acid and theother functions as a base.

Methods to determine the activity and linkage specificity of anyglycosidase are well known in the art, including a simplified HPLCprotocol (Jacob and Scudder, 1994, Methods in Enzymol. 230: 280-300). Ageneral discussion of glycosidases and glycosidase treatment is found inGlycobiology, A Practical Approach, (1993, Fukuda and Kobata eds.,Oxford University Press Inc., New York). Glycosidases useful in theinvention include, but are not limited to, sialidase, galactosidase,endoglycanase, mannosidase (i.e., α and β, ManI, ManII and ManIII,)xylosidase, fucosidase, Agrobacterium sp. β-glucosidase, Cellulomonasfungi mannosidase 2A, Humicola insolens glycosidase, Sulfolobussolfataricus glycosidase and Bacillus licheniformis glycosidase.

The choice of fucosidases for use in the invention depends on thelinkage of the fucose to other molecules. The specificities of manyα-fucosidases useful in the methods of the invention are well known tothose in the art, and many varieties of fucosidase are also commerciallyavailable (Glyko, Novato, Calif.; PROzyme, San Leandro, Calif.;Calbiochem-Novabiochem Corp., San Diego, Calif.; among others).α-Fucosidases of interest include, but are not limited to, α-fucosidasesfrom Turbo cornutus, Charonia lampas, Bacillus fulminans, Aspergillusniger, Clostridium perfringens, Bovine kidney (Glyko), chicken liver(Tyagarajan et al., 1996, Glycobiology 6:83-93) and α-fucosidase II fromXanthomonas manihotis (Glyko, PROzyme). Chicken liver fucosidase isparticularly useful for removal of core fucose from N-linked glycans.

B. Glycosyltransferases

Glycosyltransferases catalyze the addition of activated sugars (donorNDP-sugars), in a step-wise fashion, to a protein, glycopeptide, lipidor glycolipid or to the non-reducing end of a growing oligosaccharide.N-linked glycopeptides are synthesized via a transferase and alipid-linked oligosaccharide donor Do1-PP-NAG₂Glc₃Mang in an en blocktransfer followed by trimming of the core. In this case the nature ofthe “core” saccharide is somewhat different from subsequent attachments.A very large number of glycosyltransferases are known in the art.

The glycosyltransferase to be used in the present invention may be anyas long as it can utilize the modified sugar as a sugar donor. Examplesof such enzymes include Leloir pathway glycosyltransferases, such asgalactosyltransferase, N-acetylglucosaminyltransferase,N-acetylgalactosaminyltransferase, fucosyltransferase,sialyltransferase, mannosyltransferase, xylosyltransferase,glucurononyltransferase and the like.

For enzymatic saccharide syntheses that involve glycosyltransferasereactions, glycosyltransferases can be cloned, or isolated from anysource. Many cloned glycosyltransferases are known, as are theirpolynucleotide sequences. See, e.g., Taniguchi et al., 2002, Handbook ofglycosyltransferases and related genes, Springer, Tokyo.

Glycosyltransferase amino acid sequences and nucleotide sequencesencoding glycosyltransferases from which the amino acid sequences can bededuced are also found in various publicly available databases,including GenBank, Swiss-Prot, EMBL, and others.

Glycosyltransferases that can be employed in the methods of theinvention include, but are not limited to, galactosyltransferases,fucosyltransferases, glucosyltransferases,N-acetylgalactosaminyltransferases, N-acetylglucosaminyltransferases,glucuronyltransferases, sialyltransferases, mannosyltransferases,glucuronic acid transferases, galacturonic acid transferases, andoligosaccharyltransferases. Suitable glycosyltransferases include thoseobtained from eukaryotes, as well as from prokaryotes.

DNA encoding glycosyltransferases may be obtained by chemical synthesis,by screening reverse transcripts of mRNA from appropriate cells or cellline cultures, by screening genomic libraries from appropriate cells, orby combinations of these procedures. Screening of mRNA or genomic DNAmay be carried out using oligonucleotide probes generated from theglycosyltransferases nucleic acid sequence. Probes may be labeled with adetectable label, such as, but not limited to, a fluorescent group, aradioactive atom or a chemiluminescent group in accordance with knownprocedures and used in conventional hybridization assays. In thealternative, glycosyltransferases nucleic acid sequences may be obtainedby use of the polymerase chain reaction (PCR) procedure, with the PCRoligonucleotide primers being produced from the glycosyltransferasesnucleic acid sequence. See, U.S. Pat. No. 4,683,195 to Mullis et al. andU.S. Pat. No. 4,683,202 to Mullis.

A glycosyltransferases enzyme may be synthesized in a host celltransformed with a vector containing DNA encoding theglycosyltransferases enzyme. A vector is a replicable DNA construct.Vectors are used either to amplify DNA encoding the glycosyltransferasesenzyme and/or to express DNA which encodes the glycosyltransferasesenzyme. An expression vector is a replicable DNA construct in which aDNA sequence encoding the glycosyltransferases enzyme is operably linkedto suitable control sequences capable of effecting the expression of theglycosyltransferases enzyme in a suitable host. The need for suchcontrol sequences will vary depending upon the host selected and thetransformation method chosen. Generally, control sequences include atranscriptional promoter, an optional operator sequence to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and sequences which control the termination of transcription andtranslation. Amplification vectors do not require expression controldomains. All that is needed is the ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants.

1. Fucosyltransferases

In some embodiments, a glycosyltransferase used in the, method of theinvention is a fucosyltransferase. Fucosyltransferases are known tothose of skill in the art. Exemplary fucosyltransferases includeenzymes, which transfer L-fucose from GDP-fucose to a hydroxy positionof an acceptor sugar. Fucosyltransferases that transfer fromnon-nucleotide sugars to an acceptor are also of use in the presentinvention.

In some embodiments, the acceptor sugar is, for example, the GlcNAc in aGalβ(1→3,4)GlcNAcβ-group in an oligosaccharide glycoside. Suitablefucosyltransferases for this reaction include theGalβ(1→3,4)GlcNAcβ1-α(1→3,4)fucosyltransferase (FTIII E.C. No.2.4.1.65), which was first characterized from human milk (see, Palcic,et al., Carbohydrate Res. 190: 1-11 (1989); Prieels, et al., J. Biol.Chem. 256: 10456-10463 (1981); and Nunez, et al., Can. J. Chem. 59:2086-2095 (1981)) and the Galβ(1→4)GlcNAcβ-αfucosyltransferases (FTIV,FTV, FTVI) which are found in human serum. FTVII (E.C. No. 2.4.1.65), asialyl α(2→3)Galβ((1→3)GlcNAcβ fucosyltransferase, has also beencharacterized. A recombinant form of the Galβ(1→3,4)GlcNAcβ-α(1→3,4)fucosyltransferase has also been characterized (see,Dumas, et al., Bioorg. Med. Letters 1: 425-428 (1991) andKukowska-Latallo, et al., Genes and Development 4: 1288-1303 (1990)).Other exemplary fucosyltransferases include, for example, α1,2fucosyltransferase (E.C. No. 2.4.1.69). Enzymatic fucosylation can becarried out by the methods described in Mollicone, et al., Eur. J.Biochem. 191: 169-176 (1990) or U.S. Pat. No. 5,374,655.

2. Galactosyltransferases

In another group of embodiments, the glycosyltransferase is agalactosyltransferase. Exemplary galactosyltransferases include α(1,3)galactosyltransferases (E.C. No. 2.4.1.151, see, e.g., Dabkowski et al.,Transplant Proc. 25:2921 (1993) and Yamamoto et al. Nature 345: 229-233(1990), bovine (GenBank j04989, Joziasse et al., J. Biol. Chem. 264:14290-14297 (1989)), murine (GenBank m26925; Larsen et al., Proc. Nat'l.Acad. Sci. USA 86: 8227-8231 (1989)), porcine (GenBank L36152; Strahanet al., Immunogenetics 41: 101-105 (1995)). Another suitable α1,3galactosyltransferase is that which is involved in synthesis of theblood group B antigen (EC 2.4.1.37, Yamamoto et al., J. Biol. Chem. 265:1146-1151 (1990) (human)).

Also suitable for use in the methods of the invention are β(1,4)galactosyltransferases, which include, for example, EC 2.4.1.90 (LacNAcsynthetase) and EC 2.4.1.22 (lactose synthetase) (bovine (D'Agostaro etal., Eur. J. Biochem. 183: 211-217 (1989)), human (Masri et al.,Biochem. Biophys. Res. Commun. 157: 657-663 (1988)), murine (Nakazawa etal., J. Biochem. 104: 165-168 (1988)), as well as E.C. 2.4.1.38 and theceramide galactosyltransferase (EC 2.4.1.45, Stahl et al., J. Neurosci.Res. 38: 234-242 (1994)). Other suitable galactosyltransferases include,for example, α1,2 galactosyltransferases (from e.g., Schizosaccharomycespombe, Chapell et al., Mol. Biol. Cell 5: 519-528 (1994)). For furthersuitable galactosyltransferases, see Taniguchi et al. (2002, Handbook ofGlycosyltransferases and Related Genes, Springer, Tokyo), Guo et al.(2001, Glycobiology, 11(10):813-820), and Breton et al. (1998, JBiochem. 123:1000-1009).

The production of proteins such as the enzyme GalNAc T_(I-XIV) fromcloned genes by genetic engineering is well known. See, e.g., U.S. Pat.No. 4,761,371. One method involves collection of sufficient samples,then the amino acid sequence of the enzyme is determined by N-terminalsequencing. This information is then used to isolate a cDNA cloneencoding a full-length (membrane bound) transferase which uponexpression in the insect cell line Sf9 resulted in the synthesis of afully active enzyme. The acceptor specificity of the enzyme is thendetermined using a semiquantitative analysis of the amino acidssurrounding known glycosylation sites in 16 different proteins followedby in vitro glycosylation studies of synthetic peptides. This work hasdemonstrated that certain amino acid residues are overrepresented inglycosylated peptide segments and that residues in specific positionssurrounding glycosylated serine and threonine residues may have a moremarked influence on acceptor efficiency than other amino acid moieties.

3. Sialyltransferases

Sialyltransferases are another type of glycosyltransferase that isuseful in the recombinant cells and reaction mixtures of the invention.Examples of sialyltransferases that are suitable for use in the presentinvention include ST3Gal In (e.g., a rat or human ST3Gal III), ST3GalIV, ST3Gal I, ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAc I, ST6GalNAc II,and ST6GalNAc III (the sialyltransferase nomenclature used herein is asdescribed in Tsuji et al., Glycobiology 6: v-xiv (1996)). An exemplaryα(2,3)sialyltransferase referred to as α(2,3)sialyltransferase (EC2.4.99.6) transfers sialic acid to the non-reducing terminal Gal of aGalβ1→3Glc disaccharide or glycoside. See, Van den Eijnden et al., J.Biol. Chem. 256: 3159 (1981), Weinstein et al., J. Biol. Chem. 257:13845 (1982) and Wen et al., J. Biol. Chem. 267: 21011 (1992). Anotherexemplary α2,3-sialyltransferase (EC 2.4.99.4) transfers sialic acid tothe non-reducing terminal Gal of the disaccharide or glycoside. see,Rearick et al., J. Biol. Chem. 254: 4444 (1979) and Gillespie et al., J.Biol. Chem. 267: 21004 (1992). Further exemplary enzymes includeGal-β-1,4-GlcNAc α-2,6 sialyltransferase (See, Kurosawa et al. Eur. J.Biochem. 219: 375-381 (1994)).

Preferably, for glycosylation of carbohydrates of glycopeptides thesialyltransferase will be able to transfer sialic acid to the sequenceGalβ1,4GlcNAc-, Galβ1,3GlcNAc-, or Galβ1,3GalNAc-, the most commonpenultimate sequences underlying the terminal sialic acid on fullysialylated carbohydrate structures (see, Table 8).α2,8-Sialyltransferases capable of transfering sialic acid toα2,3Galβ1,4GlcNAc are also useful in the methods of the invention.

TABLE 8 Sialyltransferases which use the Galβ1,4GlcNAc sequence as anacceptor substrate Sialyl- transferase Source Sequence(s) formed Ref.ST6Gal I Mammalian NeuAcα2,6Galβ1,4GlcNAc- 1 ST3Gal III MammalianNeuAcα2,3Galβ1,4GlcNAc- 1 NeuAcα2,3Galβ1,3GlcNAc- ST3Gal IV MammalianNeuAcα2,3Galβ1,4GlcNAc- 1 NeuAcα2,3Galβ1,3GlcNAc- ST6Gal II MammalianNeuAcα2,6Galβ1,4GlcNAc- ST6Gal II Photobacterium NeuAcα2,6Galβ1,4GlcNAc-2 ST3Gal V N. meningitides NeuAcα2,3Galβ1,4GlcNAc- 3 N. gonorrhoeae 1)Goochee et al., Bio/Technology 9: 1347-1355 (1991) 2) Yamamoto et al.,J. Biochem. 120: 104-110 (1996) 3) Gilbert et al., J. Biol. Chem. 271:28271-28276 (1996)

An example of a sialyltransferase that is useful in the claimed methodsis ST3Gal III, which is also referred to as α(2,3)sialyltransferase (EC2.4.99.6). This enzyme catalyzes the transfer of sialic acid to the Galof a Galβ1,3GlcNAc or Galβ1,4GlcNAc glycoside (see, e.g., Wen et al., J.Biol. Chem. 267: 21011 (1992); Van den Eijnden et al., J. Biol. Chem.256: 3159 (1991)) and is responsible for sialylation ofasparagine-linked oligosaccharides in glycopeptides. The sialic acid islinked to a Gal with the formation of an α-linkage between the twosaccharides. Bonding (linkage) between the saccharides is between the2-position of NeuAc and the 3-position of Gal. This particular enzymecan be isolated from rat liver (Weinstein et al., J. Biol. Chem. 257:13845 (1982)); the human cDNA (Sasaki et al. (1993) J. Biol. Chem. 268:22782-22787; Kitagawa & Paulson (1994) J. Biol. Chem. 269: 1394-1401)and genomic (Kitagawa et al. (1996) J. Biol. Chem. 271: 931-938) DNAsequences are known, facilitating production of this enzyme byrecombinant expression. In a preferred embodiment, the claimedsialylation methods use a rat ST3Gal III.

An example of a sialyltransferase that is useful in the claimed methodsis CST-I from Campylobacter (see ,for example, U.S. Pat. Nos. 6,503744,6,096,529, and 6,210933 and WO99/4905 1, and published U.S. Pat.Application 2002/2,042,369). This enzyme catalyzes the transfer ofsialic acid to the Gal of a Galβ1,4Glc or Galβ1,3GalNAc. Other exemplarysialyltransferases of use in the present invention include thoseisolated from Campylobacter jejuni, including the α(2,3)sialyltransferase. See, e.g, WO99/49051.

Other sialyltransferases, including those listed in Table 8, are alsouseful in an economic and efficient large-scale process for sialylationof commercially important glycopeptides. As a simple test to find outthe utility of these other enzymes, various amounts of each enzyme(1-100 mU/mg protein) are reacted with asialo-α₁ AGP (at 1-10 mg/ml) tocompare the ability of the sialyltransferase of interest to sialylateglycopeptides relative to either bovine ST6Gal I, ST3Gal III or bothsialyltransferases. Alternatively, other glycopeptides or glycopeptides,or N-linked oligosaccharides enzymatically released from the peptidebackbone can be used in place of asialo-α₁ AGP for this evaluation.Sialyltransferases with the ability to sialylate N-linkedoligosaccharides of glycopeptides more efficiently than ST6Gal I areuseful in a practical large-scale process for peptide sialylation (asillustrated for ST3Gal III in this disclosure).

4. Other Glycosgltransferases

One of skill in the art will understand that other glycosyltransferasescan be substituted into similar transferase cycles as have beendescribed in detail for the sialyltransferase. In particular, theglycosyltransferase can also be, for instance, glucosyltransferases,e.g., Alg8 (Stagljov et al., Proc. Natl. Acad. Sci. USA 91: 5977 (1994))or Alg5 (Heesen et al., Eur. J. Biochem. 224: 71 (1994)).

N-acetylgalactosaminyltransferases are also of use in practicing thepresent invention. Suitable N-acetylgalactosaminyltransferases include,but are not limited to, α(1,3) N-acetylgalactosaminyltransferase, β(1,4)N-acetylgalactosaminyltransferases (Nagata et al., J. Biol. Chem. 267:12082-12089 (1992) and Smith et al., J. Biol Chem. 269: 15162 (1994))and peptide N-acetylgalactosaminyltransferase (Homa et al., J. Biol.Chem. 268: 12609 (1993)). Suitable N-acetylglucosaminyltransferasesinclude GnT-I (2.4.1.101, Hull et al., BBRC 176: 608 (1991)), GnT-II,GnT-III (Ihara et al., J. Biochem. 113: 692 (1993)), GnT-IV, GnT-V(Shoreibah et al., J. Biol. Chem. 268: 15381 (1993)) and GnT-VI,O-linked N-acetylglucosaminyltransferase (Bierhuizen et al., Proc. Natl.Acad. Sci. USA 89: 9326 (1992)), N-acetylglucosamine-1-phosphatetransferase (Rajput et al., Biochem J. 285: 985 (1992), and hyaluronansynthase.

Mannosyltransferases are of use to transfer modified mannose moieties.Suitable mannosyltransferases include α(1,2) mannosyltransferase, α(1,3)mannosyltransferase, α(1,6) mannosyltransferase, β(1,4)mannosyltransferase, Dol-P-Man synthase, OCh1, and Pmt1 (see, Kornfeldet al., Annu. Rev. Biochem. 54: 631-664 (1985)).

Xylosyltransferases are also useful in the present invention. See, forexample, Rodgers, et al., Biochem. J., 288:817-822 (1992); and Elbain,et al., U.S. Pat. No., 6,168,937.

Other suitable glycosyltransferase cycles are described in Ichikawa etal., JACS 114: 9283 (1992), Wong et al., J. Org. Chem. 57: 4343 (1992),and Ichikawa et al. in CARBOHYDRATES AND CARBOHYDRATE POLYMERS. Yaltami,ed. (ATL Press, 1993).

Prokaryotic glycosyltransferases are also useful in practicing theinvention. Such glycosyltransferases include enzymes involved insynthesis of lipooligosaccharides (LOS), which are produced by many gramnegative bacteria. The LOS typically have terminal glycan sequences thatmimic glycoconjugates found on the surface of human epithelial cells orin host secretions (Preston et al., Critical Reviews in Microbiology23(3): 139-180 (1996)). Such enzymes include, but are not limited to,the proteins of the rfa operons of species such as E. coli andSalmonella typhimurium, which include a β1,6 galactosyltransferase and aβ1,3 galactosyltransferase (see, e.g., EMBL Accession Nos. M80599 andM86935 (E. coli); EMBL Accession No. S56361 (S. typhimurium)), aglucosyltransferase (Swiss-Prot Accession No. P25740 (E. coli), anβ1,2-glucosyltransferase (rfaJ)(Swiss-Prot Accession No. P27129 (E.coli) and Swiss-Prot Accession No. P19817 (S. typhimurium)), and anβ1,2-N-acetylglucosaminyltransferase (rfaK)(EMBL Accession No. U00039(E. coli). Other glycosyltransferases for which amino acid sequences areknown include those that are encoded by operons such as rfaB, which havebeen characterized in organisms such as Klebsiella pneumoniae, E. coli,Salmonella typhimurium, Salmonella enterica, Yersinia enterocolitica,Mycobacterium leprosum, and the rh1 operon of Pseudomonas aeruginosa.

Also suitable for use in the present invention are glycosyltransferasesthat are involved in producing structures containinglacto-N-neotetraose,D-galactosyl-β-1,4-N-acetyl-D-glucosarninyl-P-1,3-D-galactosyl-β-1,4-D-glucose,and the Pk blood group trisaccharide sequence,D-galactosyl-α-1,4-D-galactosyl-β-1,4-D-glucose, which have beenidentified in the LOS of the mucosal pathogens Neisseria gonnorhoeae andN. meningitidis (Scholten et al., J. Med. Microbiol. 41: 236-243(1994)). The genes from N. meningitidis and N. gonorrhoeae that encodethe glycosyltransferases involved in the biosynthesis of thesestructures have been identified from N. meningitidis immunotypes L3 andL1 (Jennings et al., Mol. Microbiol. 18: 729-740 (1995)) and the N.gonorrhoeae mutant F62 (Gotshlich, J. Exp. Med. 180: 2181-2190 (1994)).In N. meningitidis, a locus consisting of three genes, lgtA, lgtB and lgE, encodes the glycosyltransferase enzymes required for addition of thelast three of the sugars in the lacto-N-neotetraose chain (Wakarchuk etal., J. Biol. Chem. 271: 19166-73 (1996)). Recently the enzymaticactivity of the lgtB and lgL4 gene product was demonstrated, providingthe first direct evidence for their proposed glycosyltransferasefunction (Wakarchuk et al., J. Biol. Chem. 271(45): 28271-276 (1996)).In N. gonorrhoeae, there are two additional genes, lgtD which addsβ-D-GalNAc to the 3 position of the terminal galactose of thelacto-N-neotetraose structure and lgtC which adds a terminal α-D-Gal tothe lactose element of a truncated LOS, thus creating the P^(k) bloodgroup antigen structure (Gotshlich (1994), supra.). In N. meniugitidis,a separate immunotype L1 also expresses the pk blood group antigen andhas been shown to carry an lgtC gene (Jennings et al., (1995), supra.).Neisseria glycosyltransferases and associated genes are also describedin U.S. Pat. No. 5,545,553 (Gotschlich). Genes forα1,2-fucosyltransferase and α1,3-fucosyltransferase from Helicobacterpylori has also been characterized (Martin et al., J. Biol. Chem. 272:21349-21356 (1997)). Also of use in the present invention are theglycosyltransferases of Campylobacter jejuni (see, Taniguchi et al.,2002, Handbook of glycosyltransferases and related genes, Springer,Tokyo).

B. Sulfotransferases

The invention also provides methods for producing peptides that includesulfated molecules, including, for example sulfated polysaccharides suchas heparin, heparan sulfate, carragenen, and related compounds. Suitablesulfotransferases include, for example, chondroitin-6-sulphotransferase(chicken cDNA described by Fukuta et al., J. Biol. Chem. 270:18575-18580 (1995); GenBank Accession No. D49915), glycosaminoglycanN-acetylglucosamine N-deacetylase/N-sulphotransferase 1 (Dixon et al.,Genomics 26: 239-241 (1995); UL18918), and glycosaminoglycanN-acetylglucosamine N-deacetylase/N-sulphotransferase 2 (murine cDNAdescribed in Orellana et al., J. Biol. Chem. 269: 2270-2276 (1994) andEriksson et al., J. Biol. Chem. 269: 10438-10443 (1994); human cDNAdescribed in GenBank Accession No. U2304).

C. Cell-Bound Glycosyltransferases

In another embodiment, the enzymes utilized in the method of theinvention are cell-bound glycosyltransferases. Although many solubleglycosyltransferases are known (see, for example, U.S. Pat. No.5,032,519), glycosyltransferases are generally in membrane-bound formwhen associated with cells. Many of the membrane-bound enzymes studiedthus far are considered to be intrinsic proteins; that is, they are notreleased from the membranes by sonication and require detergents forsolubilization. Surface glycosyltransferases have been identified on thesurfaces of vertebrate and invertebrate cells, and it has also beenrecognized that these surface transferases maintain catalytic activityunder physiological conditions. However, the more recognized function ofcell surface glycosyltransferases is for intercellular recognition(Roth, 1990, Molecular Approaches to Supracellular Phenomena,).

Methods have been developed to alter the glycosyltransferases expressedby cells. For example, Larsen et al., Proc. Natl. Acad. Sci. USA 86:8227-8231 (1989), report a genetic approach to isolate cloned cDNAsequences that determine expression of cell surface oligosaccharidestructures and their cognate glycosyltransferases. A cDNA librarygenerated from mRNA isolated from a murine cell line known to expressUDP-galactose:.β-D-galactosyl-1,4-N-acetyl-D-glucosaminideα-1,3-galactosyltransferase was transfected into COS-1 cells. Thetransfected cells were then cultured and assayed for α1-3galactosyltransferase activity.

Francisco et al., Proc. Natl. Acad. Sci. USA 89: 2713-2717 (1992),disclose a method of anchoring β-lactamase to the external surface ofEscherichia coli. A tripartite fusion consisting of (i) a signalsequence of an outer membrane protein, (ii) a membrane-spanning sectionof an outer membrane protein, and (iii) a complete mature β-lactamasesequence is produced resulting in an active surface bound β-lactamasemolecule. However, the Francisco method is limited only to prokaryoticcell systems and as recognized by the authors, requires the completetripartite fusion for proper functioning.

D. Fusion Enzymes

In other exemplary embodiments, the methods of the invention utilizefusion peptides that have more than one enzymatic activity that isinvolved in synthesis of a desired glycopeptide conjugate. The fusionpeptides can be composed of, for example, a catalytically active domainof a glycosyltransferase that is joined to a catalytically active domainof an accessory enzyme. The accessory enzyme catalytic domain can, forexample, catalyze a step in the formation of a nucleotide sugar that isa donor for the glycosyltransferase, or catalyze a reaction involved ina glycosyltransferase cycle. For example, a polynucleotide that encodesa glycosyltransferase can be joined, in-frame, to a polynucleotide thatencodes an enzyme involved in nucleotide sugar synthesis. The resultingfusion peptide can then catalyze not only the synthesis of thenucleotide sugar, but also the transfer of the sugar moiety to theacceptor molecule. The fusion peptide can be two or more cycle enzymeslinked into one expressible nucleotide sequence. In other embodimentsthe fusion peptide includes the catalytically active domains of two ormore glycosyltransferases. See, for example, U.S. Pat. No. 5,641,668.The modified glycopeptides of the present invention can be readilydesigned and manufactured utilizing various suitable fusion peptides(see, for example, PCT Patent Application PCT/CA98/01180, which waspublished as WO 99/31224 on Jun. 24, 1999.)

E. Immobilized Enzymes

In addition to cell-bound enzymes, the present invention also providesfor the use of enzymes that are immobilized on a solid and/or solublesupport. In an exemplary embodiment, there is provided aglycosyltransferase that is conjugated to a PEG via an intact glycosyllinker according to the methods of the invention. The PEG-linker-enzymeconjugate is optionally attached to solid support. The use of solidsupported enzymes in the methods of the invention simplifies the work upof the reaction mixture and purification of the reaction product, andalso enables the facile recovery of the enzyme. The glycosyltransferaseconjugate is utilized in the methods of the invention. Othercombinations of enzymes and supports will be apparent to those of skillin the art.

F. Mutagenesis of Glcosyltransferases

The novel forms of the glycosyltransferases, sialyltransferases,sulfotransferases, and any other enzymes used in the method of theinvention can be created using any of the methods described previously,as well as others well known to those in the art. Of particular interestare transferases with altered acceptor specificity and/or donorspecificity. Also of interest are enzymes with higher conversion ratesand higher stability among others.

The techniques of rational design mutagenesis can be used when thesequence of the peptide is known. Since the sequences as well as many ofthe tertiary structures of the transferases and glucosidases used in theinvention are known, these enzymes are ideal for rational design ofmutants. For example, the catalytic site of the enzyme can be mutated toalter the donor and/or acceptor specificity of the enzyme.

The extensive tertiary structural data on the glycosyltransferases andglycosidase hydrolases also make these enzyme idea for mutationsinvolving domain exchanges., Glycosyltransferases and glycosidasehydrolases are modular enzymes (see, Bourne and Henrissat, 2001, CurrentOpinion in Structural Biology 11:593-600). Glycosyltransferases aredivided into two families bases on their structure: GT-A and GT-B. Theglycosyltransferases of the GT-A family comprise two dissimilar domains,one involved in nucleotide binding and the other in acceptor binding.Thus, one could conveniently fuse the DNA sequence encoding the domainfrom one gene in frame with a domain from a second gene to create a newgene that encodes a protein with a new acceptor/donor specificity. Suchexchanges of domains could additionally include the carbohydrate modulesand other accessory domains.

The techniques of random mutation and/or directed evolution, asdescribed above, may also be used to create novel forms of theglycosyltransferases and glycosidases used in the invention.

IV. In Vitro and In Vivo Expression Systems

A. Cells for the Production of Glycopeptides

The action of glycosyltransferases is key to the glycosylation ofpeptides, thus, the difference in the expression of a set ofglycosyltransferases in any given cell type affects the pattern ofglycosylation on any given peptide produced in that cell. For a reviewof host cell dependent glycosylation of peptides, see Kabata andTakasaki, “Structure and Biosynthesis of Cell Surface Carbohydrates,” inCell Surface Carbohydrates and Cell Development, 1991, pp. 1-24, Eds.Minoru Fukuda, CRC Press, Boca Raton, Fla.

According to the present disclosure, the type of cell in which thepeptide is produced is relevant only with respect to the degree ofremodeling required to generate a peptide having desired glycosylation.For example, the number and sequence of enzymatic digestion reactionsand the number and sequence of enzymatic synthetic reactions that arerequired in vitro to generate a peptide having desired glycosylationwill vary depending on the structure of the glycan on the peptideproduced by a particular cell type. While the invention should in no waybe construed to be limited to the production of peptides from any oneparticular cell type including any cell type disclosed herein, adiscussion of several cell systems is now presented which establishesthe power of the present invention and its independence of the cell typein which the peptides are generated.

In general, and to express a peptide from a nucleic acid encoding it,the nucleic acid must be incorporated into an expression cassette,comprising a promoter element, a terminator element, and the codingsequence of the peptide operably linked between the two. The expressioncassette is then operably linked into a vector. Toward this end,adapters or linkers may be employed to join the nucleotide fragments orother manipulations may be involved to provide for convenientrestriction sites, removal of superfluous nucleotides, removal ofrestriction sites, or the like. For this purpose, in vitro mutagenesis,primer repair, restriction, annealing, resubstitutions, e.g.,transitions and transversions, may be involved. A shuttle vector has thegenetic elements necessary for replication in a cell. Some vectors maybe replicated only in prokaryotes, or may be replicated in bothprokaryotes and eukaryotes. Such a plasmid expression vector will bemaintained in one or more replication systems, preferably tworeplication systems, that allow for stable maintenance within a yeasthost cell for expression purposes, and within a prokaryotic host forcloning purposes. Many vectors with diverse characteristics are nowavailable commercially. Vectors are usually plasmids or phages, but mayalso be cosmids or mini-chromosomes. Conveniently, many commerciallyavailable vectors will have the promoter and terminator of theexpression cassette already present, and a multi-linker site where thecoding sequence for the peptide of interest can be inserted. The shuttlevector containing the expression cassette is then transformed in E. coliwhere it is replicated during cell division to generate a preparation ofvector that is sufficient to transform the host cells of the chosenexpression system. The above methodology is well know to those in theart, and protocols by which to accomplish can be found Sambrook et al.(2001, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York).

The vector, once purified from the cells in which it is amplified, isthen transformed into the cells of the expression system. The protocolfor transformation depended on the kind of the cell and the nature ofthe vector. Transformants are grown in an appropriate nutrient medium,and, where appropriate, maintained under selective pressure to insureretention of endogenous DNA. Where expression is inducible, growth canbe permitted of the yeast host to yield a high density of cells, andthen expression is induced. The secreted, mature heterologous peptidecan be harvested by any conventional means, and purified bychromatography, electrophoresis, dialysis, solvent-solvent extraction,and the like.

The techniques of molecular cloning are well-known in the art. Further,techniques for the procedures of molecular cloning can be found inSambrook et al. (2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Glover etal., (1985, DNA Cloning: A Practical Approach, Volumes I and II); Gaitet al., (1985, Oligonucleotide Synthesis); Hames and Higgins (1985,Nucleic Acid Hybridization); Hames and Higgins (1984, Transcription AndTranslation); Freshney et al., (1986, Animal Cell Culture); Perbal,(1986, Immobilized Cells And Enzymes, IRL Press); Perbal,(1984, APractical Guide To Molecular Cloning); Ausubel et al. (2002, CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc.).

B. Fungi and Yeast

Peptides produced in yeast are glycosylated and the glycan structurespresent thereon are primarily high mannose structures. In the case ofN-glycans, the glycan structures produced in yeast may contain as manyas nine or more mannose residues which may or may not contain additionalsugars added thereto. An example of the type of glycan on peptidesproduced by yeast cells is shown in FIG. 4, left side. Irrespective ofthe number of mannose residues and the type and complexity of additionalsugars added thereto, N-glycans as components of peptides produced inyeast cells comprise a trimannosyl core structure as shown in FIG. 4.When the glycan structure on a peptide produced by a yeast cell is ahigh mannose structure, it is a simple matter for the ordinary skilledartisan to remove, in vitro using available mannosidase enzymes, all ofthe mannose residues from the molecule except for those that comprisethe trimannosyl core of the glycan, thereby generating a peptide havingan elemental trimannosyl core structure attached thereto. Now, using thetechniques available in the art and armed with the present disclosure,it is a simple matter to enzymatically add, in vitro, additional sugarmoieties to the elemental trimannosyl core structure to generate apeptide having a desired glycan structure attached thereto. Similarly,when the peptide produced by the yeast cell comprises a high mannosestructure in addition to other complex sugars attached thereto, it is asimple matter to enzymatically cleave off all of the additional sugars,including extra mannose residues, to arrive at the elemental trimannosylcore structure. Once the elemental trimannosyl core structure isproduced, generation of a peptide having desired glycosylation ispossible following the directions provided herein.

By “yeast” is intended ascosporogenous yeasts (Endomycetales),basidiosporogenous yeasts, and yeast belonging to the Fungi Imperfecti(Blastomycetes). The ascosporogenous yeasts are divided into twofamilies, Spermophthoraceae and Saccharomycetaceae. The later iscomprised of four subfamilies, Schizosaccharomnycoideae (e.g., genusSchizosaccharomyces), Nadsonioideae, Lipoinycoideae, andSaccharornycoideae (e.g., genera Pichia, Kluyveronzyces, andSaccharomyces). The basidiosporogenous yeasts include the generaLeucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium, andFilobasidiella. Yeast belonging to the Fungi Imperfecti are divided intotwo families, Sporoboloinycetaceae (e.g., genera Sporoboloinyces,Bullera) and Cryptococcaceae (e.g., genus Candida). Of particularinterest to the present invention are species within the generaSaccharomyces, Pichia, Aspergillus, Trichoderma, Kluyveromyces,especially K. lactis and K. drosophiluin, Candida, Hansenula,Schizpsaccaromyces, Yarrowia, and Chlrysoporiuyn. Since theclassification of yeast may change in the future, for the purposes ofthis invention, yeast shall be defined as described in Skinner et al.,eds. 1980) Biology and Activities of Yeast (Soc. App. bacteriol. Symp.Series No. 9).

In addition to the foregoing, those of ordinary skill in the art arepresumably familiar with the biology of yeast and the manipulation ofyeast genetics. See, for example, Bacila et al., eds. (1978,Biochemistry and Genetics of Yeast, Academic Press, New York); and Roseand Harrison. (1987, The Yeasts (2nd ed.) Academic Press, London).Methods of introducing exogenous DNA into yeast hosts are well known inthe art. There are a wide variety of methods for transformation ofyeast. Spheroplast transformation is taught by Hinnen et al (1978, Proc.Natl. Acad. Sci. USA 75:1919-1933); Beggs, (1978, Nature275(5676):104-109); and Stinchcomb et al., (EPO Publication No. 45,573;herein incorporated by reference), Electroporation is taught by Beckerand Gaurante, (1991, Methods Enzymol. 194:182-187), Lithium acetate istaught by Gietz et al. (2002, Methods Enzymol. 350:87-96) and Mount etal. (1996, Methods Mol Biol. 53:139-145). For a review of transformationsystems of non-Saccharomyces yeasts, see Wang et al. (Crit RevBiotechnol. 2001;21(3):177-218). For general procedures on yeast geneticengineering, see Barr et al., (1989, Yeast genetic engineering,Butterworths, Boston).

In addition to wild-type yeast and fungal cells, there are also strainsof yeast and fungi that have been mutated and/or selected to enhance thelevel of expression of the exogenous gene, and the purity, thepost-translational processing of the resulting peptide, and the recoveryand purity of the mature peptide. Expression of an exogenous peptide mayalso be direct to the cell secretory pathway, as illustrated by theexpression of insulin (see (Kjeldsen, 2000, Appl. Microbiol. Biotechnol.54:277-286, and references cited therein). In general, to cause theexogenous peptide to be secreted from the yeast cell, secretion signalsderived from yeast genes may be used, such as those of the genes of thekiller toxin (Stark and Boyd, 1986, EMBO J. 5:1995-2002) or of the alphapheromone (Kurjan and Herskowitz, 1982, Cell 30:933; Brake et al., 1988,Yeast 4:S436).

Regarding the filamentous fungi in general, methods for geneticmanipulation can be S found in Kinghorn and Turner (1992, AppliedMolecular Genetics of Filamentous Fungi, Blackie Academic andProfessional, New York). Guidance on appropriate vectors can be found inMartinelli and Kinghom (1994, Aspergillus: 50 years, Elsevier,Amsterdam).

1. Saccharomyces

In Saccharomyces, suitable yeast vectors for use producing a peptideinclude YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76: 1035-1039,1978), YEp13 (Broach et al., Gene 8: 121-133, 1979), POT vectors(Kawasaki et al, U.S. Pat. No. 4,931,373, which is incorporated byreference herein), pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978)and derivatives thereof. Preferred promoters for use in yeast includepromoters for yeast glycolytic gene expression (Hitzeman et al., J.Biol. Chem. 255: 12073-12080, 1980; Alber and Kawasaki, J. Mol. Appl.Genet. 1: 419-434, 1982; Kawasaki, U.S. Pat. No. 4,599,311) or alcoholdehydrogenase genes (Young et al., in Genetic Engineering ofMicroorganisms for Chemicals, Hollaender et al., (eds.), p. 355, Plenum,New York, 1982; Ammerer, Meth. Enzymol. 101: 192-201, 1983), and theADH2-4^(c) promoter (Russell et al., Nature 304: 652-654, 1983; Iraniand Kilgore, U.S. patent application Ser. No. 07/784,653, CA 1,304,020and EP 284 044, which are incorporated herein by reference). Theexpression units may also include a transcriptional terminator. Apreferred transcriptional terminator is the TPI1 terminator (Alber andKawasaki, ibid.).

Examples of such yeast-bacteria shuttle vectors include Yep24 (Botsteinet al. (1979) Gene 8:17-24; pC1 (Brake et al. (1984) Proc. Natl. Acad.Sci. USA 81:4642-4646), and Yrp17 (Stnichomb et al. (1982) J. Mol. Biol.158:157). Additionally, a plasmid expression vector may be a high or lowcopy number plasmid, the copy number generally ranging from about 1 toabout 200. In the case of high copy number yeast vectors, there willgenerally be at least 10, preferably at least 20, and usually notexceeding about 150 copies of the vector in a single host. Dependingupon the heterologous peptide selected, either a high or low copy numbervector may be desirable, depending upon the effect of the vector and therecombinant peptide on the host. See, for example, Brake et al. (1984)Proc. Natl. Acad. Sci. USA 81:4642-4646. DNA constructs of the presentinvention can also be integrated into the yeast genome by an integratingvector. Examples of such vectors are known in the art. See, for example,Botstein et al. (1979) Gene 8:17-24.

The selection of suitable yeast and other microorganism hosts for thepractice of the present invention is within the skill of the art. Ofparticular interest are the Saccharomyces species S. cerevisiae, S.carlsbergensis, S. diastaticus, S. douglasli, S. kluyverin S. norbensis,and S. ovifonnis. When selecting yeast host cells for expression of adesired peptide, suitable host cells may include those shown to have,inter alia, good secretion capacity, low proteolytic activity, andoverall vigor. Yeast and other microorganisms are generally availablefrom a variety of sources, including the Yeast Genetic Stock Center,Department of Biophysics and Medical Physics, University of California,Berkeley, Calif.; and the American Type Culture Collection, Manassas Va.For a review, see Strathern et al., eds. (1981, The Molecular Biology ofthe Yeast Saccharoinyces, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.)

Methods of introducing exogenous DNA into yeast hosts are well known inthe art.

2. Pichia

The use of Pichia methanolica as a host cell for the production ofrecombinant peptides is disclosed in PCT Applications WO 97/17450, WO97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use intransforming P. methanolica are commonly prepared as double-stranded,circular plasmids, which are preferably linearized prior totransformation. For peptide production in P. methanolica, it ispreferred that the promoter and terminator in the plasmid be that of aP. methanolica gene, such as a P. methanolica alcohol utilization gene(AUG1 or AUG2). Other useful promoters include those of thedihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), andcatalase (CAT) genes, as well as those disclosed in U.S. Pat. No.5,252,726. To facilitate integration of the DNA into the hostchromosome, it is preferred to have the entire expression segment of theplasmid flanked at both ends by host DNA sequences. A preferredselectable marker for use in Pichia methanolica is a P. methanolica ADE2gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC;EC 4.1.1.21), which allows ade2 host cells to grow in the absence ofadenine. For large-scale, industrial processes where it is desirable tominimize the use of methanol, host cells in which both methanolutilization genes (AUG1 and AUG2) are deleted are preferred. Forproduction of secreted peptides, host cells deficient in vacuolarprotease genes (PEP4 and PRB1) are preferred. Electroporation is used tofacilitate the introduction of a plasmid containing DNA encoding apeptide of interest into P. methanolica cells. It is preferred totransform P. methanolica cells by electroporation using an exponentiallydecaying, pulsed electric field having a field strength of from 2.5 to4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from1 to 40 milliseconds, most preferably about 20 milliseconds. For areview of the use of Pichia pastoris for large-scale production ofantibody fragments, see Fischer et al., (1999, Biotechnol Appl Biochem.30 (Pt 2):117-120).

3. Aspergillus

Methods to express peptides in Aspergillus spp. are well known in theart, including but not limited to those described in Carrez et al.,1990, Gene 94:147-154; Contreras, 1991, Bio/Technology 9:378-381; Yeltonet al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474; Tilbum et al.,1983, Gene 26:205-221; Kelly and. Hynes, 1985, EMBO J. 4:475-479;Ballance et al., 1983, Biochem. Biophys. Res. Comm. 112:284-289; Buxtonet al., 1985, Gene 37:207-214, and U.S. Pat. No. 4,935,349, incorporatedby reference herein in its entirety. Examples of promoters useful inAspergillus are found in U.S. Pat. No. 5,252,726. Strains of Aspergillususeful for peptide expression are found in U.S. Pat. No. 4,935,349.Commercial production of exogenous peptides is available fromNovoenzymes for Aspergillus niger and Aspergillus oryzae.

4. Trichoderma

Trichoderma has certain advantages over other species of recombinanthost cells for expression of desired peptides. This organism is easy togrow in large quantities and it has the ability to glycosylate andefficiently secrete high yields of recombinant mammalian peptides intothe medium, making isolation of the peptide relatively easy. Inaddition, the glycosylation pattern on expressed peptides is moresimilar to that on human peptides than peptides expressed in many othersystems. However, there are still differences in the glycan structureson expressed peptides from these cells. For example, terminal sialicacid residues are important to the therapeutic function of a peptide ina mammalian system, since the presence of these moieties at the end ofthe glycan structure impedes peptide clearance from the mammalianbloodstream. The mechanism behind the increased biologic half-life ofsialylated molecules is believed to lie in their decreased recognitionby lectins (Drickamer, 1988, J. Biol. Chem. 263:9557-9560). However, ingeneral fungal cells do not add terminal sialic acid residues to glycanson peptides, and peptides synthesized in fungal cells are thereforeasialic. According to the present invention, this deficiency can beremedied using the in vitro glycan remodeling methods of the inventiondescribed in detail elsewhere herein.

Trichoderma species useful as hosts for the production of peptides to beremodeled include T. reesei, such as QM6a, ALK02442 or CBS383.78(Centraalbureau voor Schimmelcultures, Oosterstraat 1, PO Box 273, 3740AG Baarn, The Netherlands, or, ATCC13631 (American Type CultureCollection, Manassas Va., 10852, USA, type); T. viride (such asCBS189.79 (det. W. Gams); T. longibrachiatum, such as CBS816.68 (type);T. pseudokoningii (such as MUCL19358; Mycotheque de l'UniversiteCatholique de Louvain); T. saturnisporum CBS330.70 (type); T. harzianumCBS316.31 (det. W. Garns); T. virgatum (T. pseudokoningii) ATCC24961.Most preferably, the host is T. reesei and more preferably, it is T.reesei strains QM9414 (ATCC 26921), RUT-C-30 (ATCC 56765), and highlyproductive mutants such as VTT-D-79125, which is derived from QM9414(Nevalainen, Technical Research Centre of Finland Publications 26,(1985), Espoo, Finland).

The transformation of Trichoderma with DNA is performed using anytechnique known in the art, including that taught in European patent No.EP0244234, Harldki (1989, Bio/Technology 7:596-601) and Uusitalo (1991,J. Biotech. 17:35-50). Culture of Trichoderina is supported by previousextensive experience in industrial scale fermentation techniques; forexample, see Finkelstein, 1992, Biotechnology of Filamentous Fungi:Technology and Products, Butterworth-Heinemann, publishers, Stoneham,Mass.

5. Kluvveroinvces

Yeast belonging to the genus Kluyveromnyces have been used as hostorganisms for the production of recombinant peptides. Peptides producedby this genus of yeast are, in particular, chymosin (European Patent 96430), thaumatin (European Patent 96 910), albumin, interleukin-1β, TPA,TIMP (European Patent 361 991) and albumin derivatives having atherapeutic function (European Patent 413 622). Species of particularinterest in the genus Kluvveroinyces include K. lactis.

Methods of expressing recombinant peptides in Kluyvermyces spp. are wellknown in the art. Vectors for the expression and secretion of humanrecombinant peptides in Kluyvermyces are known in the art (Yeh, J. Cell.Biochem. Suppl. 14C:68, Abst. H402; Fleer, 1990, Yeast 6 (SpecialIssue):S449) as are procedures for transformation and expression ofrecombinant peptides (Ito et al., 1983, J. Bacteriol. 153:163-168; vanden Berg, 1990, Bio/Technology 8:135-139; U.S. Pat. No. 5,633,146,WO8304050A1, EP0096910, EP0241435, EP0301670, EP0361991, all of whichare incorporated by reference herein in their entirety). For a review ofgenetic manipulation of Kluyveromyces lactis linear DNA plasmids by genetargeting and plasmid shuffles, see Schaffrath et al. (1999, FEMSMicrobiol Lett. 178(2):201-210).

6. Chrasoporium

The fungal genus Chrysoporium has recently been used to expression offoreign recombinant peptides. A description of the proceedures by whichone of skill in the art can use Chrysoporiuin can be used to expressforeign peptides is found in WO 00/20555 (incorporated by referenceherein in its entirety). Species particularly suitable for expressionsystem include, but are not limited to, C. botryoides, C. carmichaelii,C. crassitunicatum, C. europae, C. evolceannui, F. fastidium, C.filiforme, C. gerogiae, C. globiferum, C. globiferum var. articulatum,C. globiferum var. niveum, C. hirundo, C. hispanicum, C. holmii, C.indicum, C. inops, C. keratinophilum, C. kreiselii, C. kuzurovianumn, C.ignorum, C. lobatum, C. lucknowense, C. lucklowense Garg 27K, C. medium,C. medium var. spissescens, C. mephiticum, C. merdarium, C. merdariumvar. roseumn, C. minor, C. pannicola, C. parvumn, C. parvum var.crescens, C. pilosumn, C. peodomnerderium, C. pyrifornis, C.queenslandicum, C. sigleri, C. sulfureum, C. synchronum, C. tropicum, C.undulatum, C. vallenarense, C. vespertilium, and C. zonatum.

7. Others

Methods for transforming Schwanniomnyces are disclosed in EuropeanPatent 394 538. Methods for transforming Acremonium chrysogenum aredisclosed by U.S. Pat. No. 5,162,228. Methods for transformingNeurospora are disclosed by U.S. Pat. No. 4,486,533. Also know is anexpression system specifically for Schizosaccharomyces pombe (EuropeanPatent 385 391). General methods for expressing peptides in fissionyeast, Schizosaccharomyces pombe can be found in Giga-Hama and Kumagai(1997, Foreign gene expression in fission yeast: Schizosaccharomycespombe, Springer, Berlin).

C. Mammalian Systems

As discussed above, mammalian cells typically produce a heterogeneousmixture of N-glycan structures which vary with respect to the number andarrangement of additional sugars attached to the trimannosyl core.Typically, mammalian cells produce peptides having a complex glycanstructure, such as that shown in FIG. 3, right side. Using the methodsof the present invention, a peptide produced in a mammalian cell may beremodeled in vitro to generate a peptide having desired glycosylation byfirst identifying the primary glycan structure and then determiningwhich sugars must be removed in order to remodel the glycan structure.As discussed herein, the sugars to be removed will determine whichcleavage enzymes will be used and thus, the precise steps of theremodeling process will vary depending on the primary glycan structureused as the initial substrate. A sample scheme for remodeling a glycanstructure commonly produced in mammalian cells is shown in FIG. 2. TheN-glycan biosynthetic pathway in mammalian cells has been wellcharacterized (reviewed in Moremen, 1994, Glycobiology 4:113-125). Manyof the enzymes necessary for glycan synthesis have been identified, andmutant cell lines defective in this enzymatic pathway have been isolatedincluding the Chinese hamster ovary (CHO) cell lines Lec23 (defective inalpha-glucosidase I) and Lec18 (novel GIcNAc-TVIII). The glycosylationpattern of peptides produced by these mutant cells is altered relativeto normal CHO cells. As discussed herein, the glycosylation defects inthese and other mutant cells can be exploited for the purposes ofproducing a peptide that lacks a complex glycan structure. For example,peptides produced by Lec23 cells lack sialic acid residues, and thusrequire less enzymatic manipulation in order to reduce the glycanstructure to an elemental trimannosyl core or to Man3GlcNAc4. Thus,peptides produced in these cells can serve as preferred substrates forglycan remodeling. One of ordinary skill in the art could isolate oridentify other glycosylation-defective cell lines based on knownmethods, for example the method described in Stanley et al., 1990,Somatic Cell Mol. Genet., 16: 211-223. Use of glycosylation-defectivecell lines, those identified and as yet unidentified, is included in theinvention for the purpose of generating preferred peptide substrates forthe remodeling processes described herein.

Expression vectors useful for expressing exogenous peptides in mammaliancells are numerous, and are well known to those in the art. Manymammalian expression vectors are now commercially available fromcompanies, including Novagen, Inc (Madison, Wis.), Gene Therapy Systems(San Diego, Calif.), Promega (Madison, Wis.), ClonTech Inc. (Palo Alto,Calif.), and Stratagene (La Jolla, Calif.), among others.

There are several mammalian cell lines that are particularly adept atexpressing exogenous peptides. Typically mammalian cell lines originatefrom tumor cells extracted from mammals that have become immortalized,that is to say, they can replicate in culture essentially indefinitely.These cell lines include, but are not limited to, CHO (Chinese hamsterovary, e.g. CHO-K1; ATCC No. CCL 61) and variants thereof, NSO (mousemyeloma), BNK, BHK 570 (ATCC No. CRL 10314), BHK (ATCC No. CRL 1632),Per.C6™ (immortalized human cells, Crucell N.V., Leiden, TheNetherlands), COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), HEK293, mouse L cells, T lymphoid cell lines, BW5147 cells and MDCK(Madin-Darby canine kidney), HeLa (human), A549 (human lung carcinoma),293 (ATCC No. CRL 1573; Graham et al., 1977, Gen. Virol. 36:59-72), BGMK(Buffalo Green Monkey kidney), Hep-2 (human epidermoid larynxcarcinoma), LLC-MK₂ (African Green Monkey Kidney), McCoy, NCI-H292(human pulmonary mucoepidermoid carcinoma tube), RD (rhabdomyosarcoma),Vero (African Green Monkey kidney), HEL (human embryonic lung), HumanFetal Lung-Chang, MRC5 (human embryonic lung), MRHF (human foreskin),and WI-38 (human embryonic lung). In some cases, the cells in which thetherapeutic pepfide is expressed may be cells derived from the patientto be treated, or they may be derived from another related or unrelatedmammal. For example, fibroblast cells may be isolated from the mammal'sskin tissue, and cultured and transformed in vitro. This technology iscommercially available from Transkaryotic Therapies, Inc. (Cambridge,Mass.). Almost all currently used cell lines are available from theAmerican Type Culture Collection (ATCC, Manassas, Va.) and Bio Whittaker(Walkersville, Md.).

Mammalian cells may be transformed with DNA using any one of severaltechniques that are well known to those in the art. Such techniquesinclude, but are not limited to, calcium phosphate transformation (Chenand Okayama, 1988; Graham and van der Eb, 1973; Corsaro and Pearson,1981, Somatic Cell Genetics 7:603), Diethylaminoethyl (DEAE)-dextrantransfection (Fujita et al., 1986; Lopata et al., 1984; Selden et al.,1986, ), electroporation (Neumann et al., 1982, ; Potter, 1988, ; Potteret al., 1984, ; Wong and Neuman, 1982), cationic lipid reagenttransfection (Elroy-Stein and Moss, 1990; Feigner et al., 1987; Rose etal., 1991; Whitt et al., 1990; Hawley-Nelson et al., 1993, Focus 15:73;Ciccarone et al., 1993, Focus 15:80), retroviral (Cepko et al., 1984;Miller and Baltimore, 1986; Pear et al., 1993; Austin and Cepko, 1990;Bodine et al., 1991; Fekete and Cepko, 1993; Lernischka et al., 1986;Turner et al., 1990; Williarns et al., 1984; Miller and Rosman, 1989,BioTechniques 7:980-90; Wang and Finer, 1996, Nature Med. 2:714-6),polybrene (Chaney et al, 1986; Kawai and Nishizawa, 1984),microinjection (Capecchi, 1980), and protoplast fusion (Rassoulzadeganet al., 1982; Sandri-Goldin et al., 1981; Schaffer, 1980), among others.In general, see Sambrook et al. (2001, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York) and Ausubel et al.(2002, Current Protocols in Molecular Biology, John Wiley & Sons, NewYork) for transformation techniques.

Recently the baculovirus system, popular for transformation of insectcells, has been adapted for stable transformation of mammalian cells(see, for review, Koat and Condreay, 2002, Trends Biotechnol.20:173-180, and references cited therein). The production of recombinantpeptides in cultured mammalian cells is disclosed, for example, in U.S.Pat. Nos. 4,713,339, 4,784,950; 4,579,821; and 4,656,134. Severalcompanies offer the services of transformation and culture of mammaliancells, including Cell Trends, Inc. (Middletown, Md.). Techniques forculturing mammalian cells are well known in the art, and further foundin Hauser et al. (1997, Mammalian Cell Biotechnology, Walter de Gruyer,Inc., Hawthorne, N.Y.), and Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor and references cited therein.

D. Insect

Insect cells and in particular, cultured insect cells, express peptideshaving N-linked glycan structures that are rarely sialylated and usuallycomprise mannose residues which may or may not have additional fucoseresidues attached thereto. Examples of the types of glycan structurespresent on peptides produced in cultured insect cells are shown in FIG.6, and mannose glycans thereof. In this situation, there may or may notbe a core fucose present, which if present, may be linked to the glycanvia several different linkages.

Baculovirus-mediated expression in insect cells has become particularlywell-established for the production of recombinant peptides (Altmann etal., 1999, Glycoconjugate J. 16:109-123). With regard to peptide foldingand post-translational processing, insect cells are second only tomammalian cell lines. However, as noted above, N-glycosylation ofpeptides in insect cells differs in many respects from N-glycosylationin mammalian cells particularly in that insect cells frequently generatetruncated glycan structures comprising oligosaccharides containing justthree or sometimes only two mannose residues. These structures may beadditionally substituted with fucose residues.

According to the present invention, a peptide produced in an insect cellmay be remodeled in vitro to generate a peptide with desiredglycosylation by first optionally removing any substituted fucoseresidues using an appropriate fucosidase enzyme. In instances where thepeptide comprises an elemental trimannosyl core structure following theremoval of fucose residues, then all that is required is the in vitroaddition of the appropriate sugars to the trimannosyl core structure togenerate a peptide having desired glycosylation. In instances when thepeptide might contain only two. mannose residues in the glycan structurefollowing removal of any fucose residues, a third mannose residue may beadded using a mannosyltransferase enzyme and a suitable donor moleculesuch as GDP-mannose, and thereafter the appropriate residues are addedto generate a peptide having desired glycosylation. Optionally,monoantennary glycans can also be generated from these species.

Protocols for the use of baculovirus to transform insect cells are wellknown to those in the art. Several books have been published whichprovide the procedures to use the baculovirus system to express peptidesin insect cells. These books include, but are not limited to, Richardson(Baculovirus Expression Protocols, 1998, Methods in Molecular Biology,Vol 39, Humana Pr), O'Reilly et al. (1994, Baculovirus ExpressionVectors : A Laboratory Manual, Oxford Univ Press), and King and Possee(1992, The Baculovirus Expression System: A Laboratory Guide, Chapman &Hall). In addition, there are also publications such as Lucklow (1993,Curr. Opin. Biotechnol. 4:564-572) and Miller (1993, Curr. Opin. Genet.Dev. 3:97-101).

Many patents have also been issued that related to systems forbaculoviral expression of foreign proteins. These patents include, butare not limited to, U.S. Pat. No. 6,210,966 (Culture medium for insectcells lacking glutamine and containing ammonium salt), U.S. Pat. No.6,090,584 (Use of BVACs (BaculoVirus Artificial Chromosomes) to producerecombinant peptides), U.S. Pat. No. 5,871,986 (Use of a baculovirus toexpress a recombinant nucleic acid in a mammalian cell), U.S. Pat. No.5,759,809 (Methods of expressing peptides in insect cells and methods ofkilling insects), U.S. Pat. No. 5,753,220 (Cysteine protease genedefective baculovirus, process for its production, and process for theproduction of economic peptide by using the same), U.S. Pat. No.5,750,383 (Baculovirus cloning system), U.S. Pat. No. 5,731,182(Non-mammalian DNA virus to express a recombinant nucleic acid in amammalian cell), U.S. Pat. No. 5,728,580 (Methods and culture media forinducing single cell suspension in insect cell lines), U.S. Pat. No.5,583,023 (Modified baculovirus, its preparation process and itsapplication as a gene expression vector), U.S. Pat. No. 5,571,709(Modified baculovirus and baculovirus expression vectors), U.S. Pat. No.5,521,299 (Oligonucleotides for detection of baculovirus infection),U.S. Pat. No. 5,516,657 (Baculovirus vectors for expression of secretoryand membrane-bound peptides), U.S. Pat. No. 5,475,090 (Gene encoding apeptide which enhances virus infection of host insects), U.S. Pat. No.5,472,858 (Production of recombinant peptides in insect larvae), U.S.Pat. No. 5,348,886 (Method of producing recombinant eukaryotic virusesin bacteria), U.S. Pat. No. 5,322,774 (Prokaryotic leader sequence inrecombinant baculovirus expression system), U.S. Pat. No. 5,278,050(Method to improve the efficiency of processing and secretion ofrecombinant genes in insect systems), U.S. Pat. No. 5,244,805(Baculovirus expression vectors), U.S. Pat. No. 5,229,293 (Recombinantbaculovirus), U.S. Pat. No. 5,194,376 (Baculovirus expression systemcapable of producing recombinant peptides at high levels), U.S. Pat. No.5,179,007 (Method and vector for the purification of recombinantpeptides), U.S. Pat. No. 5,169,784 (Baculovirus dual promoter expressionvector), U.S. Pat. No. 5,162,222 (Use of baculovirus early promoters forexpression of recombinant nucleic acids in stably transformed insectcells or recombinant baculoviruses), U.S. Pat. No. 5,155,037 (Insectsignal sequences usefull to improve the efficiency of processing andsecretion of recombinant nucleic acids in insect systems), U.S. Pat. No.5,147,788 (Baculovirus vectors and methods of use), U.S. Pat. No.5,110,729 (Method of producing peptides using baculovirus vectors incultured cells), U.S. Pat. No. 5,077,214 (Use of baculovirus earlypromoters for expression of recombinant genes in stably transformedinsect cells), U.S. Pat. No. 5,023,328 (Lepidopteran AKH signalsequence), and U.S. Pat. Nos. 4,879,236 and 4,745,051 (Method forproducing a recombinant baculovirus expression vector). All of theaforementioned patents are incorporated in their entirety by referenceherein.

Insect cell lines of several different species origin are currentlybeing used for peptide expression, and these lines are well known tothose in the art. Insect cell lines of interest include, but are notlimited to, dipteran and lepidopteran insect cells in general, Sf9 andvariants thereof (fall armyworm Spodoptera frugiperda), Estigmene acrea,Trichoplusia ni, Bombyx mori, Malacosoina disstri. drosophila lines Kc1and SL2 among others, and mosquito.

E. Plants

Plant cells as peptide producers present a different set of issues.While N-linked glycans produced in plants comprise a trimannosyl corestructure, this pentasaccharide backbone may comprise several differentadditional sugars as shown in FIG. 5. For example, in one instance, thetrimannosyl core structure is substituted by a β1,2 linked xyloseresidue and an α1,3 linked fucose residue. In addition, plant cells mayalso produce a Man5GlcNAc2 structure. Peptides produced in plant cellsare often highly antigenic as a result of the presence of the core α1,3fucose and xylose on the glycan structure, and are rapidly cleared fromthe blood stream when introduced into a mammal due to the absence ofterminal sialic acid residues. Therefore, unless these peptides areremodeled using the methods provided herein, they are generallyconsidered to be unsuitable as therapeutic agents in mammals. While somemonoclonal antibodies expressed in plant cells were found to benon-immunogenic in mouse, it is likely that the glycan chains were notimmunogenic because they were buried in the Pc region in theseantibodies (Chargelegue et al., 2000, Transgenic Res. 9(3):187-194).

Following the directions provided herein, it is now possible to generatea peptide produced in a plant cell wherein an increased number of theglycan structures present thereon comprise an elemental trimannosyl corestructure, or a Man3GlcNAc4 structure. This is accomplished by cleavingoff any additional sugars in vitro using a combination of appropriateglycosidases, including fucosidases, until the elemental trimannosylcore structure or the Man3GlcNAc4 structure is arrived at. Thesecleavage reactions should also include removal of any fucose or xyloseresidues from the structures in order to diminish the antigenicity ofthe fmal peptide when introduced into a mammal. Plant cells havingmutations that inhibit the addition of fucose and xylose residues to thetrimannosyl core structure are known in the art (von Schaewen et al.,1993, Plant Physiology 102:1109-1118). The use of these cells to producepeptides having glycans which lack fucose and xylose is contemplated bythe invention. Upon production of the elemental trimannosyl core orMan3GlcNAc4 structure, additional sugars may then be added thereto toarrive at a peptide having desired glycosylation that is thereforesuitable for therapeutic use in a mammal.

Transgenic plants are considered by many to be the expression system ofchoice for pharmaceutical peptides. Potentially, plants can provide acheaper source of recombinant peptides. It has been estimated that theproduction costs of recombinant peptides in plants could be between 10to 50 times lower that that of producing the same peptide in E. coli.While there are slight differences in the codon usage in plants ascompared to animals, these can be compensated for by adjusting therecombinant DNA sequences (see, Kusnadi et al., 1997, Biotechnol.Bioeng. 56:473-484; Khoudi et al., 1999, Biotechnol. Bioeng. 135-143;Hood et al., 1999, Adv. Exp. Med. Biol. 464:127-147). In addition,peptide synthesis, secretion and post-translational modification arevery similar in plants and animals, with only minor differences in plantglycosylation (see, Fischer et al., 2000, J. Biol. Regul. Homest. Agents14: 83-92). Then, products from transgenic plants are also less likelyto be contaminated by animal pathogens, microbial toxins and oncogenicsequences.

The expression of recombinant peptides in plant cells is well known inthe art. In addition to transgenic plants, peptides can also produced intransgenic plant cell cultures (Lee et al., 1997, Mol. Cell. 7:783-787),and non-transgenic plants inoculated with recombinant plant viruses.Several books have been published that describe protocols for thegenetic transformation of plant cells: Potrykus (1995, Gene transfer toplants, Springer, New York), Nickoloff (1995, Plant cell electroporationand electrofusion protocols, Humana Press, Totowa, N.Y.) and Draper(1988, Plant genetic transformation, Oxford Press, Boston).

Several methods are currently used to stably transform plant cells withrecombinant genetic material. These methods include, but are not limitedto, Agrobacterium transformation (Bechtold and Pelletier, 1998; Escuderoand Hohn, 1997; Hansen and Chilton, 1999; Touraev et al., 1997),biolistics (microprojectiles) (Finer et al., 1999; Hansen and Chilton,1999; Shilito, 1999), electroporation of protoplasts (Fromrnm et al.,1985, Ou-Lee et al., 1986; Rhodes et al., 1988; Saunders et al., 1989;Trick et al., 1997), polyethylene glycol treatment (Shilito, 1999; Tricket al., 1997), in planta mircroinjection (Leduc et al., 1996; Zhou etal., 1983), seed imbibition (Trick et al., 1997), laser beam (1996), andsilicon carbide whiskers (Thompson et al., 1995; U.S. Patent Appln. No.20020100077, incorporated by reference herein in its entirety).

Many kinds of plants are amenable to transformation and expression ofexogenous peptides. Plants of particular interest to express thepeptides to be used in the remodeling method of the invention include,but are not limited to, Arabidopsis thalliana, rapeseed (Brassica spp.;Ruiz and Blumwald, 2002, Planta 214:965-969)), soybean (Glycine max),sunflower (Helianthus unnuus), oil palm (Elaeis guineeis), groundnut(peanut, Arachis hypogaea; Deng et al., 2001, Cell. Res. 11:156-160),coconut (Cocus nucifera), castor (Ricinus communis), safflower(Carthamus tinctorius), mustard (Brassica spp. and Sinapis alba),coriander, (Coriandrum sativum), squash (Cucurbita maxima; Spencer andSnow, 2001, Heredity 86(Pt 6):694-702), linseed/flax (Linumusitatissimum; Lamblin et al., 2001, Physiol Plant 112:223-232), Brazilnut (Bertholletia excelsa), jojoba (Simmondsia chinensis), maize (Zeamays; Hood et al., 1999, Adv. Exp. Med. Biol. 464:127-147; Hood et al.,1997, Mol. Breed. 3:291-306; Petolino et al., 2000, Transgenic Research9:1-9), alfalfa (Khoudi et al., 1999, Biotechnol. Bioeng. 64:135-143),tobacco (Nicotiana tabacum; Wright et al., Transgenic Res. 10:177-181;Frigerio et al., 2000, Plant Physiol. 123:1483-1493; Cramer et al.,1996, Ann. New York Acad. Sci. 792:62-8-71; Cabanes-Macheteau et al.,1999, Glycobiology 9:365-372; Ruggiero et al., 2000, FEBS Lett.469:132-136), canola (Bai et al., 2001, Biotechnol. Prog. 17:168-174;Zhang et al., 2000, J. Anim. Sci. 78:2868-2878)), potato (Tacket et al.,1998, J. Infect. Dis. 182:302-305; Richter et al., 2000, Nat.Biotechnol. 18:1167-1171; Chong et al., 2000, Transgenic Res. 9:71-78),alfalfa (Wigdorovitz et al., 1999, Virology 255:347-353), Pea (Pisumsativum; Perrin et al., 2000, Mol. Breed. 6:345-352), rice (Oryzasativa; Stoger et al., 2000, Plant Mol. Biol. 42:583-590), cotton(Gossypium hirsutum; Kornyeyev et al., 2001, Physiol Plant 113:323-331),barley (Hordeum vulgare; Petersen et al., 2002, Plant Mol Biol49:45-58); wheat (Triticum spp.; Pellegrineschi et al., 2002, Genome45:421-430) and bean (Vicia spp.; Saalbach et al., 1994, Mol Gen Genet242:226-236).

If expression of the recombinant nucleic acid is desired in a wholeplant rather than in cultured cells, plant cells are first transformedwith DNA encoding the peptide, following which, the plant isregenerated. This involves tissue culture procedures that are typicallyoptimized for each plant species. Protocols to regenerate plants arealready well known in the art for many species. Furthermore, protocolsfor other species can be developed by one of skill in the art usingroutine experimentation. Numerous laboratory manuals are available thatdescribe procedures for plant regeneration, including but not limitedto, Smith (2000, Plant tissue culture: techniques and experiments,Academic Press, San Diego), Bhojwani and Razdan (1996, Plant tissueculture: theory and practice, Elsevier Science Pub., Amsterdan), Islam(1996, Plant tissue culture, Oxford & IBH Pub. Co., New Delhi, India),Dodds and. Roberts (1995, Experiments in plant tissue culture, New York:Cambridge University Press, Cambridge England), Bhojwani (Plant tissueculture: applications and limitations, Elsevier, Amsterdam, 1990),Trigiano and Gray (2000, Plant tissue culture concepts and laboratoryexercises,. CRC Press, Boca Raton, Fla.), and Lindsey (1991, Planttissue culture manual: fundamentals and applications, Kluwer Academic,Boston).

While purifying recombinant peptides from plants may potentially becostly, several systems have been developed to minimize these costs. Onemethod directs the synthesized peptide to the seed endosperm from whereit can easily extracted (Wright et al., 2001, Transgenic Res.10:177-181, Guda et a., 2000, Plant Cell Res. 19:257-262; and U.S. Pat.No. 5,767,379, which is incorporated by reference herein in itsentirety). An alternative approach is the co-extraction of therecombinant peptide with conventional plant products such as starch,meal or oil. In oil-seed rape, a fusion peptide of oleosin-hurudin whenexpressed in the plant, attaches to the oil body of the seed, and can beextracted from the plant seed along with the oil (Parmenter, 1995, PlantMol. Biol. 29:1167-1180; U.S. Pat. Nos. 5,650,554, 5,792,922, 5,948,682and 6,288,304, and US application 2002/0037303, all of which areincorporated in their entirely by reference herein). In a variation onthis approach, the oleosin is fused to a peptide having affinity for theexogenous co-expressed peptide of interest (U.S. Pat. No. 5,856,452,incorporated by reference herein in its entirety).

Expression of recombinant peptides in plant plastids, such as thechloroplast, generates peptides having no glycan structures attachedthereto, similar to the situation in prokaryotes. However, the yield ofsuch peptides is vastly greater when expressed in these plant cellorganelles, and thus this type of expression system may have advantagesover other systems. For a general review on the technology for plastidexpression of exogenous peptides in higher plants, see Hager and Beck(2000, AppI. Microbiol. Biotechnol. 54:302-310, and references citedtherein). Plastid expression has been particularly successful in tobacco(see, for example, Staub et al., 2000, Nat. Biotechnol. 18:333-338).

F. Transgenic Animals

Introduction of a recombinant DNA into the fertilized egg of an animal(e.g., a mammal) may be accomplished using any number of standardtechniques in transgenic animal technology. See, e.g., Hogan et al.,Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986; and U.S. Pat. No.5,811,634, which is incorporated by reference herein in its entirety.Most commonly, the recombinant DNA is introduced into the embryo by wayof pronuclear microinjection (Gordon et al., 1980, PNAS 77:7380-7384;Gordon and Ruddle, 1981, Science 214:1244-1246; Brinster et al., 1981,Cell 27:223-231; Costantini and Lacy, 1981, Nature 294:92-94).Microinjection has the advantage of being applicable to a wide varietyof species. Preimplantation embryos may also be transformed withretroviruses (Jaenisch and Mintz, 1974, Proc. Natl. Acad. Sci. U.S.A.71:1250-1254; Jaenisch et al., 1976, Hamatol Bluttransfus. 19:341-356;Stuhlmann et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:7151-7155).Retroviral mediated transformation has the advantage of adding singlecopies of the recombinant nucleic acid to the cell, but it produces ahigh degree of mosaicism. Most recently, embryonic stem cell-mediatedtechniques have been used (Gossler et al., 1986, Proc. Natl. Acad. Sci.U.S.A. 83:9065-9069), transfer of entire chromosomal segments (Lavitranoet al., 1989, Cell 57:717-723), and gamete transfection in conjunctionwith in vitro fertilization (Lavitrano et al., 1989, Cell 57:717-723)have also been used. Several books of laboratory procedures have beenpublished disclosing these techniques: Cid-Arregui and García-Carrancá(1998, Microinjection and Transgenesis: Strategies and Protocols,Springer, Berlin), Clarke (2002, Transgenesis Techniques: Principles andProtocols, Humana Press, Totowa, N.J.), and Pinkert (1994, TransgenicAnimal Technology: A Laboratory Handbook, Academic Press, San Diego).

Once the recombinant DNA is introduced into the egg, the egg isincubated for a short period of time and is then transferred into apseudopregnant animal of the same species from which the egg wasobtained (Hogan et al., supra). In the case of mammals, typically 125eggs are injected per experiment, approximately two-thirds of which willsurvive the procedure. Twenty viable eggs are transferred into apseudopregnant mammal, four to ten of which will develop into liveprogeny. Typically, 10-30% of the progeny (in the case of mice) carrythe recombinant DNA.

While the entire animal can be used as an expression system for thepeptides of the invention, in a preferred embodiment, the exogenouspeptide accumulates in products of the animal, from which it can beharvested without injury to the animal. In preferred embodiments, theexogenous peptide accumulates in milk, eggs, hair, blood, and urine.

If the recombinant peptide is to be accumulated in the milk of theanimal, suitable mammals are ruminants, ungulates, domesticated mammals,and dairy animals. Particularly preferred animals are goats, sheep,camels, cows, pigs, horses, oxen, and llamas. Methods for generatingtransgenic cows that accumulate a recombinant peptide in their milk arewell known: see, Newton (1999, J. Immunol. Methods 231:159-167), Ebertet al. (1991, Biotechnology 9: 835-838), and U.S. Pat. Nos. 6,210,736,5,849,992, 5,843,705, 5,827,690, 6,222,094, all of which areincorporated herein by reference in their entirety. The generation oftransgenic mammals that produce a desired recombinant peptide iscommercially available from GTC Biotherapeutics, Framingham, Mass.

If the recombinant peptide is to be accumulated in eggs, suitable birdsinclude, but are not limited to, chickens, geese, and turkeys. Otheranimals of interest include, but are not limited to, other species ofavians, fish, reptiles and amphibians. The introduction of recombinantDNA to a chicken by retroviral transformation is well known in the art:Thoraval et al. (1995, Transgenic Research 4:369-376), Bosselman et al.,(1989, Science 243: 533-535), Petropoulos et al. (1992, J. Virol. 66:3391-3397), U.S. Pat. No. 5,162,215, incorporated by reference herein inits entirety. Successful transformation of chickens with recombinant DNAalso been achieved wherein DNA is introduced into blastodermal cells andblastodermal cells so transfected are introduced into the embryo:Brazolot et al. (1991, Mol. Reprod. Dev. 30: 304312), Fraser, et al.(1993, Int. J. Dev. Biol. 37: 381-385), and Petitte et al. (1990,Development 108: 185-189). High throughput technology has been developedto assess whether a transgenic chicken expresses the desired peptide(Harvey et al., 2002, Poult. Sci. 81:202-212, U.S. Pat. No. 6,423,488,incorporated by reference herein in its entirety). Using retroviraltransformation of chicken with a recombinant DNA, exogenousbeta-lactamase was accumulated in the egg white of the chicken (Harveyet al., 2002, Nat. Biotechnol. 20(4):396-399). The production ofchickens producing exogenous peptides in egg is commercially availablefrom AviGenics, Inc., Athens Ga.

G. Bacteria

Recombinantly expressed peptides produced in bacteria are not generallyglycosylated. However, bacteria systems capable of glycosylatingpeptides are becoming evident and therefore it is likely thatglycosylated recombinant peptides may be produced in bacteria in thefuture.

Numerous bacterial expression systems are known in the art. Preferredbacterial species include, but are not limited to, E. coli and Bacillusspecies. The expression of recombinant peptides in E. coli is well knownin the art. Protocols for E. coli-based expression systems are found inU.S. Appln No. 20020064835, U.S. Pat. Nos. 6,245,539, 5,606,031,5,420,027, 5,151,511, and RE33,653, among others. Methods to transformbacteria include, but are not limited to, calcium chloride (Cohen etal., 1972, Proc. Natl. Acad. Sci. U.S.A. 69:2110-2114; Hanahan, 1983, J.Mol. Biol. 166:557-580; Mandel and Higa, 1970, J. Mol. Biol. 53:159-162)and electroporation (Shigekawa and Dower, 1988, Biotechniques6:742-751), and those described in Sambrook et al., 2001 (supra). For areview of laboratory protocols on microbial transformation andexpression systems, see Saunders and Saunders (1987, Microbial GeneticsApplied to Biotechnology: Principles and Techniques of Gene Transfer andManipulation, Croom Helm, London), Pühler (1993, Genetic Engineering ofMicroorganisms, Weinheim, New York), Lee et al., (1999, MetabolicEngineering, Marcel Dekker, New York), Adolph (1996, Microbial GenomeMethods, CRC Press, Boca Raton), and Birren and Lai (1996, NonmammalianGenomic Analysis : A Practical Guide, Academic Press, San Diego),

For a general review on the literature for peptide expression in E. colisee Balbas (2001, Mol. Biotechnol. 19:251-267). Several companies nowoffer bacterial strains selected for the expression of mammalianpeptides, such as the Rosetta™ strains of E. coli (Novagen, inc.,Madison, Wis.; with enhanced expression of eukaryotic codons notnormally used in bacteria cells, and enhanced disulfide bond formation),

H. Cell Engineering

It will be apparent from the present disclosure that the more uniformthe starting material produced by a cell, the more efficient will be thegeneration in vitro of large quantities of peptides having desiredglycosylation. Thus, the genetic engineering of host cells to produceuniformly glycosylated peptides as starting material for the in vitroenzymatic reactions disclosed herein, provides a significant advantageover using a peptide starting material having a heterogeneous set ofglycan structures attached thereto. One preferred peptide startingmaterial for use in the present invention is a peptide having primarilyglycan molecules which consist solely of an elemental trimannosyl corestructure. Another preferred starting material is Man3GlcNAc4. Followingthe remodeling process, the preferred peptides will give rise to thegreatest amount of peptides having desired glycosylation, and thusimproved clinical efficacy. However, other glycan starting material isalso suitable for use in the methods described herein, in that forexample, high mannose glycans may be easily reduced, in vitro, toelemental trimannosyl core structures using a series of mannosidases. Asdescribed elsewhere herein, other glycan starting material may also beused, provided it is possible to cleave off all extraneous sugarmoieties so that the elemental trimannosyl core structure or Man3GlcNAc4is generated. Thus, the purpose of using genetically engineered cellsfor the production of the peptides of the present invention is togenerate peptides having as uniform as possible a glycan structureattached thereto, wherein the glycan structure can be remodeled in vitroto generate a peptide having desired glycosylation. This will result ina dramatic reduction in production costs of these peptides. Since theglycopeptides produced using this methodology will predominantly havethe same N-linked glycan structure, the post-production modificationprotocol can be standardized and optimized to produce a greaterbatch-to-batch consistency of fmal product. As a result, the finalcompleted-chain products may be less heterogeneous than those presentlyavailable. The products will have an improved biological half-life andbioactivity as compared to the products of the prior art. Alternatively,if desired, the invention can be used to introduce limited and specificheterogeneity, e.g., by choosing reaction conditions that result indifferential addition of sugar moieties.

Preferably, though not as a rigid requirement, the geneticallyengineered cell is one which produces peptides having glycan structurescomprised primarily of an elemental trimannosyl core structure orMan3GlcNAc4. At a minimum, the proportion of these preferred structuresproduced by the genetically engineered cell must be enough to yield apeptide having desired glycosylation following the remodeling protocol.

In general, any eukaryotic cell type can be modified to become a hostcell of the present invention. First, the glycosylation pattern of bothendogenous and recombinant glycopeptides produced by the organism aredetermined in order to identify suitable additions/deletions ofenzymatic activities that result in the production of elementaltrimannosyl core glycopeptides or Man3GlcNAc4 glycopeptides. This willtypically entail deleting activities that use trimannosyl glycopeptidesas substrates for a glycosyltransferase reaction and inserting enzymaticactivities that degrade more complex N-linked glycans to produce shorterchains. In addition, genetically engineered cells may produce highmannose glycans, which may be cleaved by mannosidase to produce desiredstarting glycan structures. The mannosidase may be active in vivo in thecell (i.e., the cell may be genetically engineered to produce them), orthey may be used in in vitro post production reactions.

Techniques for genetically modifying host cells to alter theglycosylation profile of expressed peptides are well-known. See, e.g.,Altmann et al. (1999, Glycoconjugate J. 16: 109-123), Ailor et al.(2000, Glycobiology 10(8): 837-847), Jarvis et al., (In vitrogenConference, March, 1999, abstract), Hollister and Jarvis, (2001,Glycobiology 11(1): 1-9), and Palacpac et al., (1999, PNAS USA 96:4697), Jarvis et al., (1998. Curr. Opin. Biotechnol. 9:528-533),Gerngross (U.S. Patent Publication No. 20020137134), all of whichdisclose techniques to “mammalianize”πinsect or plant cell expressionsystems by transfecting insect or plant cells with glycosyltransferasegenes.

Techniques also exist to genetically alter the glycosylation profile ofpeptides expressed in E. coli. E. coli has been engineered with variousglycosyltransferases from the bacteria Neisseria meningitidis andAzorhizobium to produce oligosaccharides in vivo (Bettler et al., 1999,Glycoconj. 3. 16:205-212). E. coli which has been genetically engineeredto over-express Neisseria meningitidis β1,3 N acetylglucosaminyltransferase IgtA gene will efficiently glycosylate exogenouslactose (Priem et al., 2002, Glycobiology 12:235-240).

Fungal cells have also been genetically modified to produce exogenousglycosyltransferases (Yoshida et al., 1999, Glycobiology, 9(l):53-58;Kalsner et al., 1995, Glycoconj. J. 12:360-370; Schwientek and Ernst,1994, Gene 145(2):299-303; Chiba et al, 1995, Biochem J. 308:405-409).

Thus, in one aspect, the present invention provides a cell thatglycosylates a glycopeptide population such that a proportion ofglycopeptides produced thereby have an elemental trimannosyl core or aMan3GlcNAc4 structure. Preferably, the cell produces a peptide having aglycan structure comprised solely of an elemental trimannosyl core. At aminimum, the proportion of peptides having an elemental trimannosyl coreor a Man3GIcNAc4 structure is enough to yield peptides having desiredglycosylation following the remodeling process. The cell has introducedinto it one or more heterologous nucleic acid expression units, each ofwhich may comprise one or more nucleic acid sequences encoding one ormore peptides of interest. The natural form of the glycopeptide ofinterest may comprise one or more complex N-linked glycans or may simplybe a high mannose glycan.

The cell may be any type of cell and is preferably a eukaryotic cell.The cell may be a mammalian cell such as human, mouse, rat, rabbit,hamster or other type of mammalian cell. When the cell is a mammaliancell, the mammalian cell may be derived from or contained within anon-human transgenic mammal where the cell in the mammal encodes thedesired glycopeptide and a variety of glycosylating and glycosidaseenzymes as necessary for the production of desired glycopeptidemolecules. In addition, the cell may be a fungal cell, preferably, ayeast cell, or the cell may be an insect or a plant cell. Similarly,when the cell is a plant cell, the plant cell may be derived from orcontained within a transgenic plant, wherein the plant encodes thedesired glycopeptide and a variety of glycosylating and glycosidaseenzymes as are necessary for the production of desired glycopeptidemolecules.

In some embodiments the host cell may be a eukaryotic cell expressingone or more heterologous glycosyltransferase enzymes and/or one or moreheterologous glycosidase enzymes, wherein expression of a recombinantglycopeptide in the host cell results in the production of a recombinantglycopeptide having an elemental trimannosyl core as the primary glycanstructure attached thereto.

In some embodiments the heterologous glycosyltransferase enzyme usefulin the cell may be selected from a group consisting of any knownglycosyltransferase enzyme included for example, in the list ofGlycosyltransferase Families available in Taniguchi et al. (2002,Handbook of Glycosyltransferases and Related Genes, Springer, New York).

In other embodiments, the heterologous glycosylase enzyme may beselected from a group consisting of mannosidase 1, mannosidase 2,mannosidase 3, and other mannosidases, including, but not limited to,microbial mannosidases. Additional disclosure regarding enzymes usefulin the present invention is provided elsewhere herein.

In yet other embodiments, the host cell may be a eukaryotic cell whereinone or more endogenous glycosyltransferase enzymes and/or one or moreendogenous glycosidase enzymes have been inactivated such thatexpression of a recombinant glycopeptide in the host cell results in theproduction of a recombinant glycopeptide having an elemental trimannosylcore as the primary glycan structure attached thereto.

In additional embodiments, the host cell may express heterologousglycosyltransferase enzymes and/or glycosidase enzymes while at the sametime one or more endogenous glycosyltransferase enzymes and/orglycosidase enzymes are inactivated. Endogenous glycosyltransferaseenzymes and/or glycosidase enzymes may be inactivated using anytechnique known to those skilled in the art including, but not limitedto, antisense techniques and techniques involving insertion of nucleicacids into the genome of the host cell. In some embodiments, theendogenous enzymes may be selected from a group consisting of GnT-I, aselection of mannosidases, xylosyltransferase, core α1,3fucosyltransferase, serine/threonine O-mannosyltransferases, and thelike.

Alternatively, an expression system that naturally glycosylates peptidessuch that the N-linked glycans are predominantly the trimannosyl coretype, or the Man3GlcNAc4 type, can be exploited. An example of a celltype that produces the trimannosyl core is Sf9 cells. Other-suchexpression systems can be identified by analyzing glycopeptides that arenaturally or recombinantly expressed in cells and selecting those whichexhibit the desired glycosylation characteristics. The invention shouldbe construed to include any and all such cells for the production of thepeptides of the present invention.

V. Purification of Glycan Remodeled and/or Glycoconjugated Peptides

If the modified glycoprotein is produced intracellularly or secreted, asa first step, the particulate debris, either host cells, lysedfragments, is removed, for example, by centrifugation orultrafiltration; optionally, the protein may be concentrated with acommercially available protein concentration filter, followed byseparating the peptide variant from other impurities by one or moresteps selected from immunoaffinity chromatography, ion-exchange columnfractionation (e.g., on diethylaminoethyl (DEAE) or matrices containingcarboxymethyl or sulfopropyl groups), chromatography on Blue-Sepharose,CM Blue-Sepharose, MONO-Q, MONO-S, lentil lectin-Sepharose,WGA-Sepharose, Con A-Sepharose, Ether Toyopearl, Butyl Toyopearl, PhenylToyopearl, or protein A Sepharose, SDS-PAGE chromatography, silicachromatography, chromatofocusing, reverse phase HPLC (RP-HPLC), gelfiltration using, e.g., Sephadex molecular sieve or size-exclusionchromatography, chromatography on colurns that selectively bind thepeptide, and ethanol, pH or ammonium sulfate precipitation, membranefiltration and various techniques.

Modified peptides produced in culture are usually isolated by initialextraction from cells, enzymes, etc., followed by one or moreconcentration, salting-out, aqueous ion-exchange, or size-exclusionchromatography steps. Additionally, the modified glycoprotein may bepurified by affinity chromatography. Then, HPLC may be employed forfinal purification steps.

A protease inhibitor, e.g., phenylmethylsulfonylfluoride (PMSF) may beincluded in any of the foregoing steps to inhibit proteolysis andantibiotics may be included to prevent the growth of adventitiouscontaminants.

Within another embodiment, supernatants from systems which produce themodified peptide of the invention are first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate may be applied to a suitablepurification matrix. For example, a suitable affinity matrix maycomprise a ligand for the peptide, a lectin or antibody molecule boundto a suitable support. Alternatively, an anion-exchange resin may beemployed, for example, a matrix or substrate having pendant DEAE groups.Suitable matrices include acrylamide, agarose, dextran, cellulose, orother types commnonly employed in protein purification. Alternatively, acation-exchange step may be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Sulfopropyl groups are particularly preferred.

Then, one or more RP-HPLC steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, may beemployed to further purify a peptide variant composition. Some or all ofthe foregoing purification steps, in various combinations, can also beemployed to provide a homogeneous modified glycoprotein.

The modified peptide of the invention resulting from a large-scalefermentation may be purified by methods analogous to those disclosed byUrdal et aL, J Chromatog. 296: 171 (1984). This reference describes twosequential, RP-HPLC steps for purification of recombinant human IL-2 ona preparative HPLC column. Alternatively, techniques such as affinitychromatography may be utilized to purify the modified glycoprotein.

VI. Preferred Peptides and Nucleic Acids Encoding Preferred Peptides

The present invention includes isolated nucleic acids encoding variouspeptides and proteins, and similar molecules or fragments thereof. Theinvention should not be construed to be limited in any way solely to theuse of these peptides in the.methods of the invention, but rather shouldbe construed to include any and all peptides presently available orwhich become available to those in the art. In addition, the inventionshould not be construed to include only one particular nucleic acid oramino acid sequence for the peptides listed herein, but rather should beconstrued to include any and all variants, homologs, mutants, etc. ofeach of the peptides. It should be noted that when a particular peptideis identified as having a mutation or other alteration in the sequencefor that peptide, the numbering of the amino acids which identify thealteration or mutation is set so that the first amino acid in the maturepeptide sequence is amino acid no. 1, unless otherwise stated herein.

Preferred peptides include, but are not limited to human granulocytecolony stimulating factor (G-CSF), human interferon alpha (IFN-alpha),human interferon beta (IFN-beta), human Factor VII (Factor VII), humanFactor IX (Factor IX), human follicle stimulating hormone (FSH), humanerythropoietin (EPO), human granulocyte/macrophage colony stimulatingfactor (GM-CSF), human interferon gamma (IFN-gamrna), humanalpha-1-protease inhibitor (also known as alpha-1-antitrypsin oralpha-1-trypsin inhibitor; A-1-PI), glucocerebrosidase, humantissue-type activator (TPA), human interleukin-2 (IL-2), human FactorVIII (Factor VIR), a 75 kDa tumor necrosis factor receptor fused to ahuman IgG immunoglobulin Fc portion, commercially known as ENBREL™ orETANERCEPT™ (chimeric TNFR), human urokinase (urokinase), a Fab fragmentof the human/mouse chimeric monoclonal antibody that specifically bindsglycoprotein IIb/IIIa and the vitronectin alphav beta₃ receptor, knowncommercially as REOPRO™ or ABCIXIMAB (chimeric anti-glycoproteinIIb/IIIa), a mouse/human chimeric monoclonal antibody that specificallybinds human HER2, known commercially as HERCEPTIN™ (chimeric anti-HER2),a human/mouse chimeric antibody that specifically binds the A antigenicsite or the F protein of respiratory syncytial virus commercially knownas SYNAGIS™ or PALIVIZUMAB (chimeric anti-RSV), a chimeric human/mousemonoclonal antibody that specifically binds CD20 on human B-cells, knowncommercially as RITUXAN™ or RITUXAMAB (chimeric anti-CD20), humanrecombinant DNase (DNase), a chimeric human/mouse monoclonal antibodythat specifically binds human tumor necrosis factor, known commerciallyas REMICADE™ or INFLIXIMAB (chimeric anti-TNF), human insulin, thesurface antigen of a hepatitis B virus (adw subtype; HBsAg), and humangrowth hormone (HGH), alpha-galactosidase A (Fabryzyme™), α-Iduronidase(Aldurazyme™), antithrombin (antithrombin III, AT-III), human chorionicgonadotropin (hCG), interferon omega, and the like.

The isolated nucleic acid of the invention should be construed toinclude an RNA or a DNA sequence encoding any of the above-identifiedpeptides of the invention, and any modified forms thereof, includingchemical modifications of the DNA or RNA which render the nucleotidesequence more stable when it is cell free or when it is associated witha cell. As a non-limiting example, oligonucleotides which contain atleast one phosphorothioate modification are known to confer upon theoligonucleotide enhanced resistance to nucleases. Specific examples ofmodified oligonucleotides include those which contain phosphorothioate,phosphotriester, methyl phosphonate, short chain alkyl or cycloalkylintersugar linkages, or short chain heteroatomic or heterocyclicintersugar (“backbone”) linkages. In addition, oligonucleotides havingmorpholino backbone structures (U.S. Pat. No. 5,034,506) or polyamidebackbone structures (Nielsen et al., 1991, Science 254: 1497) may alsobe used.

Chemical modifications of nucleotides may also be used to enhance theefficiency with which a nucleotide sequence is taken up by a cell or theefficiency with which it is expressed in a cell. Any and allcombinations of modifications of the nucleotide sequences arecontemplated in the present invention.

The present invention should not be construed as being limited solely tothe nucleic and amino acid sequences disclosed herein. As described inmore detail elsewhere herein, once armed with the present invention, itis readily apparent to one skilled in the art that other nucleic acidsencoding the peptides of the present invention can be obtained byfollowing the procedures described herein (e.g., site-directedmutagenesis, frame shift mutations, and the like), and procedures thatare well-known in the art.

Also included are isolated nucleic acids encoding fragments of peptides,wherein the peptide fragments retain the desired biological activity ofthe peptide. In addition, although exemplary nucleic acids encodingpreferred peptides are disclosed herein in relation to specific SEQ IDNOS, the invention should in no way be construed to be limited to anyspecific nucleic acid disclosed herein. Rather, the invention should beconstrued to include any and all nucleic acid molecules having asufficient percent identity with the sequences disclosed herein suchthat these nucleic acids also encode a peptide having the desiredbiological activity disclosed herein. Also contemplated are isolatednucleic acids that are shorter than full length nucleic acids, whereinthe biological activity of the peptide encoded thereby is retained.Methods to determine the percent identity between one nucleic acid andanother are disclosed elsewhere herein as are assays for thedetermination of the biological activity of any specific preferredpeptide.

Also as disclosed elsewhere herein, any other number of procedures maybe used for the generation of derivative, mutant, or variant forms ofthe peptides of the present invention using recombinant DNA methodologywell known in the art such as, for example, that described in Sambrooket al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, New York) and Ausubel et al. (1997, Current Protocolsin Molecular Biology, Green & Wiley, New York). Procedures for theintroduction of amino acid changes in a peptide or polypeptide byaltering the DNA sequence encoding the peptide are well known in the artand are also described in Sambrook et al. (1989, supra); Ausubel et al.(1997, supra).

The invention includes a nucleic acid encoding a G-CSF, IFN-alpha,IFN-beta, Factor VII, Factor IX, FSH, EPO, GM-CSF, IFN-gamma, A-1-PI,glucocerebrosidase, TPA, IL-2, Factor VIII, chimeric TNFR, urokinase,chimeric anti-glycoprotein IIb/IIa, chimeric anti-HER2, chimericanti-RSV, chimeric anti-CD20, DNase, chimeric anti-TNF, human insulin,HBsAg, and HGH, wherein a nucleic acid encoding a tag peptide iscovalently linked thereto. That is, the invention encompasses a chimericnucleic acid wherein the nucleic acid sequence encoding a tag peptide iscovalently linked to the nucleic acid encoding a peptide of the presentinvention. Such tag peptides are well known in the art and include, forinstance, green fluorescent protein (GFP), myc, myc-pyruvate kinase(myc-PK), His₆, maltose binding protein (MBP), an influenza virushemagglutinin tag polypeptide, a flag tag polypeptide (FLAG), and aglutathione-S-transferase (GST) tag polypeptide. However, the inventionshould in no way be construed to be limited to the nucleic acidsencoding the above-listed tag peptides. Rather, any nucleic acidsequence encoding a peptide which may fumction in a manner substantiallysimilar to these tag peptides should be construed to be included in thepresent invention.

The nucleic acid comprising a nucleic acid encoding a tag peptide can beused to localize a peptide of the present invention within a cell, atissue, and/or a whole organism (e.g., a mammalian embryo), detect apeptide of the present invention secreted from a cell, and to study therole(s) of the peptide in a cell. Further, addition of a tag peptidefacilitates isolation and purification of the “tagged” peptide such thatthe peptides of the invention can be produced and purified readily.

The invention includes the following preferred isolated peptides: G-CSF,IFN-alpha, IFN-beta, Factor VII, Factor IX, FSH, EPO, GM-CSF,IFN-garnma, A-1-PI, glucocerebrosidase, TPA, IL-2, Factor VIII, chimericTNFR, urokinase, chimeric anti-glycoprotein IIb/IIIa, chimericanti-HER2, chimeric anti-RSV, chimeric anti-CD20, DNase, chimericanti-TNF, human insulin, HBsAg, HGH, alpha-galactosidase A,α-Iduronidase, antithrombin III, hCG, and interferon omega, and thelike.

The present invention should also be construed to encompass“derivatives,” “mutants”, and “variants” of the peptides of theinvention (or of the DNA encoding the same) which derivatives, mutants,and variants are peptides which are altered in one or more amino acids(or, when referring to the nucleotide sequence encoding the same, arealtered in one or more base pairs) such that the resulting peptide (orDNA) is not identical to the sequences recited herein, but has the samebiological property as the peptides disclosed herein, in that thepeptide has biological/biochemical properties of G-CSF, IFN-alpha,IFN-beta, Factor VII, Factor IX, FSH, EPO, GM-CSF, IFN-gamma, A-1-PI,glucocerebrosidase, TPA, IL-2, Factor VIII, chimeric TNFR, urokinase,chimeric anti-glycoprotein IIb/IIIa, chimeric anti-HER2, chimericanti-RSV, chimeric anti-CD20, DNase, chimeric anti-TNF, human insulin,HBsAg, and HGH.

Further included are fragments of peptides that retain the desiredbiological activity of the peptide irrespective of the length of thepeptide. It is well within the skill of the artisan to isolate smallerthan full length forms of any of the peptides useful in the invention,and to determine, using the assays provided herein, which isolatedfragments retain a desired biological activity and are therefore usefulpeptides in the invention.

A biological property of a protein of the present invention should beconstrued to include, but not be limited to include the ability of thepeptide to function in the biological assay and environments describedherein, such as reduction of inflammation, elicitation of an immuneresponse, blood-clotting, increased hematopoietic output, proteaseinhibition, immune system modulation, binding an antigen, growth,alleviation of treatment of a disease, DNA cleavage, and the like.

A. G-CSF

The present invention encompasses a method for the modification of theglycan structure on G-CSF. G-CSF is well known in the art as a cytokineproduced by activated T-cells, macrophages, endothelial cells, andstromal fibroblasts. G-CSF primarily acts on the bone marrow to increasethe production of inflammatory leukocytes, and further functions as anendocrine hormone to initiate the replenishment of neutrophils consumedduring inflammatory functions. G-CSF also has clinical applications inbone marrow replacement following chemotherapy.

A remodeled G-CSF peptide may be administered to a patient selected fromthe group consisting of a non-myeloid cancer patient receivingmyelosuppressive chemotherapy, a patient having Acute Myeloid Leukemia(AML) receiving induction or consolidation chemotherapy, a non-myeloidcancer patient receiving a bone marrow transplant, a patient undergoingperipheral blood progenitor cell collection, a patient having severechronic neutropenia, and a patient having persistent neutropenia andalso having advanced HIV infection. Preferably, the patient is a humanpatient.

While G-CSF has been shown to be an important and useful compound fortherapeutic applications in mammals, especially humans, present methodsfor the production of G-CSF from recombinant cells results in a producthaving a relatively short biological life, an inaccurate glycosylationpattern that could potentially lead to immunogenicity, loss of function,and an increased need for both larger and more frequent doses in orderto achieve the same effect, and the like.

G-CSF has been isolated and cloned, the nucleic acid and amino acidsequences of which are presented as SEQ ID NO:1 and SEQ ID NO:2,respectively (FIGS. 58A and 58B, respectively). The present inventionencompasses a method for modifying G-CSF, particularly as it relates tothe ability of G-CSF to function as a potent and functional biologicalmolecule. The skilled artisan, when equipped with the present disclosureand the teachings herein, will readily understand that the presentinvention provides compositions and methods for the modification ofG-CSF.

The present invention further encompasses G-CSF variants, as well knownin the art. As an example, but in no way meant to be limiting to thepresent invention, a G-CSF variant has been described in U.S. Pat. No.6,166,183, in which a G-CSF comprising the natural complement of lysineresidues and further linked to one or two polyethylene glycol moleculesis described. Additionally, U.S. Pat. Nos. 6,004,548, 5,580,755,5,582,823, and 5,676,941 describe a G-CSF variant in which one or moreof the cysteine residues at position 17, 36, 42, 64, and 74 are replacedby alanine or alternatively serine. U.S. Pat. No. 5,416,195 describes aG-CSF molecule in which the cysteine at position 17, the aspartic acidat position 27, and the serines at positions 65 and 66 are substitutedwith serine, serine, proline, and proline, respectively. Other variantsare well known in the art, and are described in, for example, U.S. Pat.No. 5,399,345.

The expression and activity of a modified G-CSF molecule of the presentinvention can be assayed using methods well known in the art, and asdescribed in, for example, U.S. Pat. No. 4,810,643. As an example,activity can be measured using radio-labeled thymidine uptake assays.Briefly, human bone marrow from healthy donors is subjected to a densitycut with Ficoll-Hypaque (1.077 g/ml, Pharmacia, Piscataway, N.J.) andlow density cells are suspended in Iscove's medium (GIBCO, La Jolla,Calif.) containing 10% fetal bovine serum, glutamine and antibiotics.About 2×10⁴ human bone marrow cells are incubated with either controlmedium or the G-CSF or the present invention in 96-well flat bottomplates at about 37° C. in 5% CO₂ in air for about 2 days. Cultures arethen pulsed for about 4 hours with 0.5 μCi/well of ³H-thymidine (NewEngland Nuclear, Boston, Mass.) and uptake is measured as described in,for example, Ventua, et al.(1983, Blood 61:781). An increase in³H-thymidine incorporation into human bone marrow cells as compared tobone marrow cells treated with a control compound is an indication of aactive and viable G-CSF compound.

B. IFN alpha, IFN beta and IFN omega

The present invention further encompasses a method for the remodelingand modification of IFN alpha, IFN beta and IFN omega. IFN alpha is partof a family of approximately twenty peptides of approximately 18 kDa inweight. IFN omega is very similar in structure and function to IFNalpha. IFN omega is useful for treatment of hepatitis C virus infectionwhen an immune response to IFN alpha is mounted in the host renderingthat treatment ineffective. Antibodies raised against IFN alpha do notcross-react with IFN omega. Thus, treatment of hepatitis C may continueusing IFN omega when IFN alpha therapy is no longer possible.

IFN alpha, omega, and IFN beta, collectively known as the Type Iinterferons, bind to the same cellular receptor and elicit similarresponses. Type I IFNs inhibit viral replication, increase the lyticpotential of NK cells, modulate MHC molecule expression, and inhibitcellular proliferation, among other things. Type I IFN has been used asa therapy for viral infections, particularly hepatitis viruses, and as atherapy for multiple sclerosis.

Current compositions of Type I IFN are, as described above, usefulcompounds for both the modulation of aberrant irnnunological responsesand as a therapy for a variety of diseases. However, they are hamperedby decreased potency and function, and a limited half-life in the bodyas compared to natural cytokines comprising the natural complement ofglycosylation.

A remodeled interferon-alpha peptide may be administered to a patientselected from the group consisting of a patient having hairy cellleukemia, a patient having malignant melanoma, a patient havingfollicular lymphoma, a patient having condylomata acuminata, a patienthaving AIDS-related Kaposi's sarcoma, a patient having Hepatitis C, apatient having Hepatitis B, a patient having a human papilloma virusinfection, a patient having Chronic Myeloid Leukemia (CML), a patienthaving chronic phase Philadelphia chromosome (Ph) positive ChronicMyelogenous Leukemia, a patient having non-Hodgkin's lymphoma (NHL), apatient having lymphoma, a patient having bladder cancer, and a patienthaving renal cancer. Preferably, the patient is a human patient.

A remodeled interferon-beta peptide may be administered to a patientselected from the group consisting of a patient having multiplesclerosis (MS), a patient having Hepatitis B, a patient having HepatitisC, a patient having human papilloma virus infection, a patient havingbreast cancer, a patient having brain cancer, a patient havingcolorectal cancer, a patient having pulmonary fibrosis, and a patienthaving rheumatoid arthritis. Preferably, the patient is a human patient.

A remodeled interferon-omega peptide may be admninistered to a patientselected from the group consisting of a patient having hairy cellleukemia, a patient having malignant melanoma, a patient havingfollicular lymphoma, a patient having condylomata acuminata, a patienthaving AIDS-related Kaposi's sarcoma, a patient having Hepatitis C, apatient having Hepatitis B, a patient having a human papilloma virusinfection, a patient having Chronic Myeloid Leukemia (CML), a patienthaving chronic phase Philadelphia chromosome (Ph) positive ChronicMyelogenous Leukemia, a patient having non-Hodgkin's lymphoma (NHL), apatient having lymphoma, a patient having bladder cancer, and a patienthaving renal cancer. Preferably, the patient is a human patient.

The prototype nucleotide and amino acid sequence for IFN alpha is setforth herein as SEQ ID NO:3 and SEQ ID NO:4, respectively (FIGS. 59A and59B, respectively). The prototype nucleotide and amino acid sequence forIFN omega is set forth herein as SEQ ID NO:74 and SEQ ID NO:75,respectively (FIGS. 59C and 59D, respectively). IFN beta comprises asingle gene product of approximately 20 kDa, the nucleic acid and aminoacid sequence of which are presented herein as SEQ ID NO:5 and SEQ IDNO:6 (FIGS. 60A and 60B, respectively). The present invention is notlimited to the nucleotide and amino acid sequences herein. One of skillin the art will readily appreciate that many variants of IFN alpha existboth naturally and as engineered derivatives. Similarly, IFN beta hasbeen modified in attempts to achieve a more beneficial therapeuticprofile. Examples of modified Type I IFNs are well known in the art (seeTable 9), and are described in, for example U.S. Pat. No. 6,323,006, inwhich cysteine-60 is substituted for tyrosine, U.S. Pat. Nos. 4,737,462,4,588,585, 5,545,723, and 6,127,332 where an IFN beta with asubstitution of a variety of amino acids is described. Additionally,U.S. Pat. Nos. 4,966,843, 5,376,567, 5,795,779 describe IFN alpha-61 andIFN-alpha-76. U.S. Patent Nos .4,748,233 and 4,695,543 describe IFNalpha gx-1, whereas U.S. Pat. No. 4,975,276 describes IFN alpha-54. Inaddition, U.S. Pat. Nos. 4,695,623, 4,897,471, 5,661,009, and 5,541,293all describe a consensus IFN alpha sequence to represent all variantsknown at the date of filing. While this list of Type I IFNs and variantsthereof is in no way meant to be exhaustive, one of skill in the artwill readily understand that the present invention encompasses IFN betaand IFN alpha molecules, derivatives, and variants known or to bediscovered in the future.

TABLE 9 Interferon-α Isoforms. α type AA characteristic  1a A¹¹⁴  1bV¹¹⁴  2a K²³-H³⁴  2b R²³-H³⁴  2c R²³-R³⁴  4a A⁵¹-E¹¹⁴  4b T⁵¹-V¹¹⁴  7aM¹³²-K¹⁵⁹-G¹⁶¹  7b M¹³²-Q¹⁵⁹-R¹⁶¹  7c T¹³²-K¹⁵⁹-G¹⁶¹  8aV⁹⁸-L⁹⁹-C¹⁰⁰-D¹⁰¹-R¹⁶¹  8b S⁹⁸-C⁹⁹-V¹⁰⁰-M¹⁰¹-R¹⁶¹  8cS⁹⁸-C⁹⁹-V¹⁰⁰-M¹⁰¹-D¹⁶¹ Δ(162-166) 10a S⁸-L⁸⁹ 10b T⁸-I⁸⁹ 14aF¹⁵²-Q¹⁵⁹-R¹⁶¹ 14b F¹⁵²-K¹⁵⁹-G¹⁶¹ 14c L¹⁵²-Q¹⁵⁹-R¹⁶¹ 17a P³⁴-S⁵⁵-I¹⁶¹17b H³⁴-S⁵⁵-I¹⁶¹ 17c H³⁴-S⁵⁵-R¹⁶¹ 17d H³⁴-P⁵⁵-R¹⁶¹ 21a M⁹⁶ 21b L⁹⁶

Methods of expressing IFN in recombinant cells are well known in theart, and is easily accomplished using techniques described in, forexample U.S. Pat. No. 4,966,843, and in Sambrook et al. (2001, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NewYork) and Ausubel et al. (1997, Current Protocols in Molecular Biology,Green & Wiley, New York). Assays to determine the biological activity ofa Type I IFN modified by the present invention will be well known to theskilled artisan. For example, the assay described in Rubinstein et al.,(1981, Journal of Virology 37:755-758) is commonly used to determine theeffect of an Type I IFN by measuring the cytopathic effects of viralinfection on a population of cells. This method is only one of manyknown in the art for assaying the biological function of a Type IFN.

C. Factor VIIa

The present invention further encompasses a method for the remodelingand modification of Factor VII. The blood coagulation pathway is acomplex reaction comprising many events. An intermediate event in thispathway is Factor VII, a proenzyme that participates in the extrinsicpathway of blood coagulation by converting (upon its activation toFactor VIIa) Factor X to Xa in the presence of tissue factor and calciumions. Factor Xa in tarn then converts prothrombin to thrombin in thepresence of Factor Va, calcium ions and phospholipid. The activation ofFactor X to Factor Xa is an event shared by both the intrinsic andextrinsic blood coagulation pathways, and therefore, Factor VIIa can beused for the treatment of patients with deficiencies or inhibitors ofFactor VIII. There is also evidence to suggest that Factor VIIa mayparticipate in the intrinsic pathway as well therefore increasing theprominence and importance of the role of Factor VII in bloodcoagulation.

Factor VII is a single-chain glycoprotein with a molecular weight ofapproximately 50 kDa. In this form, the factor circulates in the bloodas an inactive zymogen. Activation of Factor VII to VIIa may becatalyzed by several different plasma proteases, such as Factor XIIa.Activation of Factor VII results in the formation of a heavy chain and alight chain held together by at least one disulfide bond. Further,modified Factor VII molecules that cannot be converted to Factor VIIahave been described, and are useful as anti-coagulation remedies, suchas in the case of blood clots, thrombosis, and the like. Given theimportance of Factor VII in the blood coagulation pathway, and its useas a treatment for both increased and decreased levels of coagulation,it follows that a molecule that has a longer biological half-life,increased potency, and in general, a therapeutic profile more similar towild-type Factor VII as it is synthesized and secreted in the healthyhuman would be beneficial and useful as a treatment for bloodcoagulation disorders.

A remodeled Factor VII peptide may be administered to a patient selectedfrom the group consisting of a hemophiliac patient having a bleedingepisode, a patient having Hemophilia A, a patient with Hemophilia B, apatient having Hemophilia A, wherein the patient also has antibodies toFactor VIII, a patient having Hemophilia B, wherein the patient also hasantibodies to Factor IX, a patient having liver cirrhosis, a cirrhoticpatient having an orthotopic liver transplant, a cirrhotic patienthaving upper gastrointestinal bleeding, a patient having a bone marrowtransplant, and a patient having a liver resection. Preferably, thepatient is a human patient.

Factor VII has been cloned and sequenced, and the nucleic acid and aminoacid sequences are presented herein as SEQ ID NO:7 and SEQ ID NO:8(FIGS. 61A and 61B, respectively). The present invention should in noway be construed as limited to the Factor VII nucleic acid and aminoacid sequences set forth herein. Variants of Factor VII are describedin, for example, U.S. Pat. Nos. 4,784,950 and 5,580,560, in whichlysine-38, lysine-32, arginine-290, arginine-341, isoleucine-42,tyrosine-278, and tyrosine-332 is replaced by a variety of amino acids.Further, U.S. Pat. Nos. 5,861,374, 6,039,944, 5,833,982, 5,788,965,6,183,743, 5,997,864, and 5,817,788 describe Factor VII variants thatare not cleaved to form Factor VIIa. The skilled artisan will recognizethat the blood coagulation pathway and the role of Factor VII thereinare well known, and therefore many variants, both naturally occurringand engineered, as described above, are included in the presentinvention.

Methods for the expression and to determine the activity of Factor VIIare well known in the art, and are described in, for example, U.S. Pat.No. 4,784,950. Briefly, expression of Factor VII, or variants thereof,can be accomplished in a variety of both prokaryotic and eukaryoticsystems, including E. coli, CHO cells, BHK cells, insect cells using abaculovirus expression system, all of which are well known in the art.

Assays for the activity of a modified Factor VII prepared according tothe methods of the present invention can be accomplished using methodswell known in the art. As a non-limiting example, Quick et al.(Hemorragic Disease and Thrombosis, 2nd ed., Leat Febiger, Philadelphia,1966), describes a one-stage clotting assay useful for determining thebiological activity of a Factor VII molecule prepared according to themethods of the present invention.

D. Factor IX

The present invention further encompasses a method for remodeling and/ormodifing Factor IX. As described above, Factor IX is vital in the bloodcoagulation cascade. A deficiency of Factor IX in the body characterizesa type of hemophilia (type B). Treatment of this disease is usuallylimited to intravenous tranfusion of human plasma protein concentratesof Factor IX. However, in addition to the practical disadvantages oftime and expense, transfusion of blood concentrates involves the risk oftransmission of viral hepatitis, acquired immune deficiency syndrome orthromboembolic diseases to the recipient.

While Factor IX has demonstrated itself as an important and usefulcompound for therapeutic applications, present methods for theproduction of Factor IX from recombinant cells (U.S. Pat. No. 4,770,999)results in a product with a rather short biological life, an inaccurateglycosylation pattern that could potentially lead to immunogenicity,loss of function, an increased need for both larger and more frequentdoses in order to achieve the same effect, and the like.

A remodeled Factor IX peptide may be administered to a patient selectedfrom the group consisting of a hemophiliac patient having a bleedingepisode and also having Hemophilia B, a patient having Hemophilia B, apatient having Hemophilia B and having antibodies to Factor IX, apatient having liver cirrhosis, a cirrhotic patient having an orthotopicliver transplant, a cirrhotic patient having upper gastrointestinalbleeding, a patient having a bone marrow transplant, and a patienthaving a liver resection. A remodeled Factor IX peptide may also beadministered to control and/or prevent hemorrhagic episodes in a patienthaving Hemophilia B, congenital Factor IX deficiency, or Christmasdisease. A remodeled Factor IX peptide may also be administered to apatient to control and/or prevent hemorrhagic episodes in the patientduring surgery. Preferably, the patient is a human patient.

The nucleic and amino acid sequences of Factor IX is set forth herein asSEQ ID NO:9 and SEQ ID NO:10 (FIGS. 62A and 62B, respectively). Thepresent invention is in no way limited to the sequences set forthherein. Factor IX variants are well known in the art, as described in,for example, U.S. Pat. Nos. 4,770,999, 5,521,070 in which a tyrosine isreplaced by an alanine in the first position, U.S. Pat. No. 6,037,452,in which Factor XI is linked to an alkylene oxide group, and U.S. Pat.No. 6,046,380, in which the DNA encoding Factor IX is modified in atleast one splice site. As demonstrated herein, variants of Factor IX arewell known in the art, and the present disclosure encompasses thosevariants known or to be developed or discovered in the future.

Methods for determining the activity of a modified Factor IX preparedaccording to the methods of the present invention can be carried outusing the methods described above, or additionally, using methods wellknown in the art, such as a one stage activated partial thromboplastintime assay as described in, for example, Biggs (1972, Human BloodCoagulation Haemostasis and Thrombosis (Ed. 1), Oxford, Blackwell,Scientific, pg. 614). Briefly, to assay the biological activity of aFactor IX molecule developed according to the methods of the presentinvention, the assay can be performed with equal volumes of activatedpartial thromboplastin reagent, Factor IX deficient plasma isolated froma patient with hemophilia B using sterile phlebotomy techniques wellknown in the art, and normal pooled plasma as standard, or the sample.In this assay, one unit of activity is defined as that amount present inone milliliter of normal pooled plasma. Further, an assay for biologicalactivity based on the ability of Factor IX to reduce the clotting timeof plasma from Factor IX-deficient patients to normal can be performedas described in, for example, Proctor and Rapaport (1961, Amer. J. Clin.Path. 36: 212).

E. FSH

The present invention further includes a method for remodeling and/ormodifying FSH. Human reproductive function is controlled in part by afamily of heterodimeric human glycoprotein hormones which have a common92 amino acid glycoprotein alpha subunit, but differ in theirhormone-specific beta subunits. The family includes follicle-stimulatinghormone (FSH), luteinizing hormone (LH), thyrotropin orthyroid-stimulating hormone (TSH), and human chorionic gonadotropin(hCG). Human FSH and LH are used therapeutically to regulate variousaspects of metabolism pertinent to reproduction in the human female. Forexample, FSH partially purified from urine is used clinically tostimulate follicular maturation in anovulatory women with anovulatorysyndrome or luteal phase deficiency. Luteinizing hormone (LH) and FSHare used in combination to stimulate the development of ovarianfollicles for in vitro fertilization. The role of FSH in thereproductive cycle is sufficiently well-known to permit therapeutic use,but difficulties have been encountered due, in part, to theheterogeneity and impurity of the preparation from native sources. Thisheterogeneity is due to variations in glycosylation pattern.

FSH is a valuable tool in both in vitro fertilization and stimulation offertilization in vivo, but as stated above, its clinical efficacy hasbeen hampered by inconsistency in glycosylation of the protein. Ittherefore seems apparent that a method for remodeling FSH will be ofgreat benefit to the reproductive sciences.

A remodeled FSH peptide may be administered to a patient selected fromthe group consisting of a patient undergoing intrauterine insemination(IUI), a patient undergoing in vitro fertilization (IVF), and aninfertile patient. A remodeled FSH peptide may also be administered toinduce or increase ovulation in a patient, to stimulate development ofan ovarian follicle in a patient, to induce gametogenic follicle growthin a patient, to stimulate, induce or increase follicle development andsubsequent ovulation in a patient, or to treat infertility in a patient.Preferably, the patient is a human female patient. A remodeled FSHpeptide may also be administered to a patient having a pituitarydeficiency or to a patient during puberty. Preferably this patient is ahuman male patient.

FSH has been cloned and sequenced, the nucleic and amino acid sequencesof which are presented herein as SEQ ID NO:11, SEQ ID NO: 12,respectively (alpha subunit) and SEQ ID NO:13 and SEQ ID NO:14,respectively (beta subunit) (FIGS. 63A, 63B, 63C and 63D, respectively).The skilled artisan will readily appreciate that the present inventionis not limited to the sequences depicted herein, as variants of FSH arewell known in the art. As a non-limiting example, U.S. Pat. No.5,639,640 describes the beta subunit comprising two different amino acidsequences and U.S. Pat. No. 5,338,835 describes a beta subunitcomprising an additional amino acid sequence of approximatelytwenty-seven amino acids derived from the beta subunit of humanchorionic gonadotropin. Therefore, the present invention comprises FSHvariants, both natural and engineered by the human hand, all well knownin the art.

Methods to express FSH in cells, both prokaryotic and eukaryotic, arewell known in the art and abundantly described in the literature (U.S.Pat. Nos. 4,840,896, 4,923,805, 5,156,957). Further, methods forevaluating the biological activity of a remodeled FSH molecule of thepresent invention are well known in the art, and are described in, forexample, U.S. Pat. No. 4,589,402, in which methods for determining theeffect of FSH on fertility, egg production, and pregnancy rates isdescribed in both non-human primates and human subjects.

F. EPO

The present invention further comprises a method of remodeling and/ormodifying EPO. EPO is an acidic glycoprotein of approximately 34 kDa andmay occur in three natural forms: alpha, beta, and asialo. The alpha andbeta forms differ slightly in carbohydrate components but have the samepotency, biological activity and molecular weight. The asialo form is analpha or beta form with the terminal sialic acid removed. EPO is presentin very low concentrations in plasma when the body is in a healthy statewherein tissues receive sufficient oxygenation from the existing numberof erythrocytes. This normal concentration is enough to stimulatereplacement of red blood cells which are lost normally through aging.The amount of erythropoietin in the circulation is increased under,conditions of hypoxia when oxygen transport by blood cells in thecirculation is reduced. Hypoxia may be caused by loss of large amountsof blood through hemorrhage, destruction of red blood cells byover-exposure to radiation, reduction in oxygen intake due to highaltitudes or prolonged unconsciousness, or various forms of anemia.Therefore EPO is a useful compound for replenishing red blood cellsafter radiation therapy, anemia, and other life-threatening conditions.

A remodeled EPO peptide may be administered to a patient selected fromthe group consisting of a patient having anemia, an anemic patienthaving chronic renal insufficiency, an anemic patient having end stagerenal disease, an anemic patient undergoing dialysis, an anemic patienthaving chronic renal failure, an anemic Zidovudine-treated HIV infectedpatient, an anemic patient having non-myeloid cancer and undergoingchemotherapy, and an anemic patient scheduled to undergo non-cardiac,non-vascular surgery. A remodeled EPO peptide may also be administeredto a patient undergoing surgery to reduce the need for an allogenicblood transfusion. A remodeled EPO peptide may also be administered to apatient at increased risk for a perioperative blood transfusion withsignificant anticipated blood loss. Preferably, the patient is a humanpatient.

In light of the importance of EPO in aiding in the recovery from avariety of diseases and disorders, the present invention is useful forthe production of EPO with a natural, and therefore more effectivesaccharide component. EPO, as it is currently synthesized, lacks thefull glycosylation complement, and must therefore be administered morefrequently and in higher doses due to its short life in the body. Theinvention also provides for the production of PEGylated EPO moleculeswith greatly improved half-life compared with what might be achieved bymaximizing desirable glycoforms.

EPO has been cloned and sequenced, and the nucleotide and amino acidsequences are present herein as SEQ ID NO:15 and SEQ ID NO:16,respectively (FIGS. 64A and 64B, respectively). It will be readilyunderstood by one of skill in the art that the sequences set forthherein are only an example of the sequences encoding and comprising EPO.As an example, U.S. Pat. No. 6,187,564 describes a fusion proteincomprising the amino acid sequence of two or more EPO peptides, U.S.Pat. Nos. 6,048,971 and 5,614,184 describe mutant EPO molecules havingamino acid substitutions at positions 101, 103, 104, and 108. U.S. Pat.No. 5,106,954 describes a truncated EPO molecule, and U.S. Pat. No.5,888,772 describes an EPO analog with substitutions at position 33,139, and 166. Therefore, the skilled artisan will realize that thepresent invention encompasses EPO and EPO derivatives and variants asare well documented in the literature and art as a whole.

Additionally, methods of expressing EPO in a cell are well known in theart. As exemplified in U.S. Pat. Nos. 4,703,008, 5,688,679, and6,376,218, among others, EPO can be expressed in prokaryotic andeukaryotic expression systems. Methods for assaying the biologicalactivity of EPO are equally well known in the art. As an example, theKrystal assay (Krystal, 1983, Exp. Hematol. 11:649-660) can be employedto determine the activity of EPO prepared according to the methods ofthe present invention. Briefly, the assay measures the effect oferythropoietin on intact mouse spleen cells. Mice are treated withphenylhydrazine to stimulate production of erythropoietin-responsive redblood cell progenitor cells. After treatment, the spleens are removed,intact spleen cells are isolated and incubated with various amounts ofwild-type erythropoietin or the erythropoietin proteins describedherein. After an overnight incubation, ³H-thymidine is added and itsincorporation into cellular DNA is measured. The amount of ³H-thymidineincorporation is indicative of erythropoietin-stimulated production ofred blood cells via interaction of erythropoietin with its cellularreceptor. The concentration of the erythropoietin protein of the presentinvention, as well as the concentration of wild-type erythropoietin, isquantified by competitive radioimmunoassay methods well known in theart. Specific activities are calculated as international units measuredin the Krystal assay divided by micrograms as measured asimmunoprecipitable protein by radioimmunoassay.

Several different mutated EPO's with different glycosylation patternshave been reported. Many have improved stimulation of reticulocytosisactivity without effecting the half-life of the peptide in the bloodstream of the animal. It is contemplated that mutated EPO peptides canbe used in place of the native EPO peptides in any of the glycanremodeling, glycoPEGylation and/or glycoconjugation embodimentsdescribed herein. Preferred mutations of EPO are listed in the followingtable, but not limited to those listed in the table (see, for example,Chern et al., 1991, Eur. J. Biochem. 202:225-229; Grodberg et al., 1993,Eur. J. Biochem. 218:597-601; Burns et al., 2002, Blood 99:4400-4405;U.S. Pat. No. 5,614,184; GenBank Accession No. AAN76993; O'Connell etal., 1992, J. Biol. Chem. 267:25010-25018; Elliott et al., 1984, Proc.Natl. Acad. Sci. U.S.A. 81:2708-2712; Biossel et al., 1993, J. Biol.Chem. 268:15983-15993). The most preferred mutations of EPO are Arg¹³⁹to Ala¹³⁹, Arg¹⁴³ to Ala¹⁴³ and Lys¹⁵⁴ to Ala¹⁵⁴. The preferred nativeEPO from which to make these mutants is the 165 aa form, which isdepicted in FIG. 65; however other native forms of EPO may also be used.Finally, the mutations described in Table 10 may be combined with eachother and with other mutations to make EPO peptides that are useful inthe present invention.

TABLE 10 Mutations of EPO. Mutation Citation Notes Arg¹³⁹ to Ala¹³⁹ J.Biol. Chem. 269: 22839 Increased activity in bioassays of 120% to (1994)150%. Arg¹⁴³ to Ala¹⁴³ Increased activity in bioassays than native EPO.Lys¹⁵⁴ to Ala¹⁵⁴ J. Biol. Chem. 269: 22839 Increased activity inbioassays of 120% to (1994) 150%. Ser¹²⁶ to Met¹²⁶ Met⁵⁴ to Leu⁵⁴ U.S.Pat. No. 4,385,260 Met⁵⁴ to Leu⁵⁴ U.S. Pat. No. 4,385,260 Asn³⁸ to Gln³⁸Δ1-30 Funakoshi et al., 1993, Mutant isolated from hepatocellularSer¹³¹Leu¹³² to Biochem. Biophys. Res. carcinoma. Asn¹³¹Phe¹³² Commun.195: 717-722. Pro¹⁴⁹ to Gln¹⁴⁹ Genbank Accession No. AAD13964. Gly¹⁰¹ toAla¹⁰¹ U.S. Pat. No. 5,615,184 Increased activity in bioassays of 120%to J. Biol. Chem. 269: 22839 150%. (1994) Ser¹⁴⁷ to Ala¹⁴⁷ Wen et al.,1994, J. Biol. Mutation results in increased bioactivity. and/or Chem.269: 22839-22846. Ile¹⁴⁶ to Ala¹⁴⁶ Ser¹²⁶ to Thr¹²⁶ J. Biol. Chem. 267:25010 (1992)

G. GM-CSF

The present invention encompasses a method for the modification ofGM-CSF. GM-CSF is well known in the art as a cytokine produced byactivated T-cells, macrophages, endothelial cells, and stromalfibroblasts. GM-CSF primarily acts on the bone marrow to increase theproduction of inflammatory leukocytes, and further functions as anendocrine hormone to initiate the replenishment of neutrophils consumedduring inflammatory functions. Further GM-CSF is a macrophage-activatingfactor and promotes the differentiation of Lagerhans cells intodendritic cells. Like G-CSF, GM-CSF also has clinical applications inbone marrow replacement following chemotherapy.

While G-CSF has demonstrated itself as an important and useful compoundfor therapeutic applications, present methods for the production ofG-CSF from recombinant cells results in a product with a rather shortbiological life, an inaccurate glycosylation pattern that couldpotentially lead to immunogenicity, loss of function, an increased needfor both larger and more frequent doses in order to achieve the sameeffect, and the like.

A remodeled GM-CSF peptide may be administered to a patient selectedfrom the group consisting of a patient having Acute Myelogenous Leukemia(AML) or acute non-lymphocytic leukemia (ANLL), a patient undergoingleukapheresis to collect hematopoietic progenitor cells from theperipheral blood, a patient undergoing transplantation of autologousperipheral blood progenitor cells, a non-Hodgkin's lymphoma (NHL)patient undergoing an autologous bone marrow transplant, a Hodgkin'sdisease patient undergoing an autologous bone marrow transplant, and anacute lymphoblastic leukemia (ALL) patient undergoing an autologous bonemarrow transplant. A remodeled GM-CSF peptide may also be administeredto a patient to accelerate myeloid engraftment, to shorten time toneutrophil recovery following chemotherapy, to mobilize hematopoieticprogenitor cells into the peripheral blood for collection byleukapheresis, or to promote myeloid reconstitution after autologous orallogeneic bone marrow transplantation (BMT). A remodeled GM-CSF peptidemay also be administered to a patient in which bone marrowtransplantation has failed or in which myeloid engraftment is delayed.Preferably, the patient is a human patient.

GM-CSF has been isolated and cloned, the nucleic acid and amino acidsequences of which are presented as SEQ ID NO:17 and SEQ ID NO:18,respectively (FIGS. 66A and 66B, respectively). The present inventionencompasses a method for modifying GM-CSF, particularly as it relates tothe ability of GM-CSF to function as a potent and functional biologicalmolecule. The skilled artisan, when equipped with the present disclosureand the teachings herein, will readily understand that the presentinvention provides compositions and methods for the modification ofGM-CSF.

The present invention further encompasses GM-CSF variants, as well knownin the art. As an example, but in no way meant to be limiting to thepresent invention, a GM-CSF variant has been described in WO 86/06358,where the protein is modified for an alternative quaternary structure.Further, U.S. Pat. No. 6,287,557 describes a GM-CSF nucleic acidsequence ligated into the genome of a herpesvirus for gene therapyapplications. Additionally, European Patent Publication No. 0288809(corresponding to PCT Patent Publication No. WO 87/02060) reports afusion protein comprising IL-2 and GM-CSF. The IL-2 sequence can be ateither the N— or C-terminal end of the GM-CSF such that after acidcleavage of the fusion protein, GM-CSF having either N— or C-terminalsequence modifications can be generated. Therefore, GM-CSF derivatives,mutants, and variants are well known in the art, and are encompassedwithin the methods of the present invention.

The expression and activity of a modified GM-CSF molecule of the presentinvention can be assayed using methods well known in the art, and asdescribed in, for example, U.S. Pat. No. 4,810,643. As an example,activity can be measured using radio-labeled thymidine uptake assays.Briefly, human bone marrow from healthy donors is subjected to a densitycut with Ficoll-Hypaque (1.077 g/ml, Pharmacia, Piscataway, N.J.) andlow density cells are suspended in Iscove's medium (GIBCO, La Jolla,Calif.) containing 10% fetal bovine serum, glutamine and antibiotics.About 2×10⁴ human bone marrow cells are incubated with either controlmedium or the GM-CSF or the present invention in 96-well flat bottomplates at about 37° C. in 5% CO₂ in air for about 2 days. Cultures arethen pulsed for about 4 hours with 0.5 μCi/well of ³H-thymidine (NewEngland Nuclear, Boston, Mass.) and uptake is measured as described in,for example, Ventua, et al.(1983, Blood 61:781). An increase in³H-thymidine incorporation into human bone marrow cells as compared tobone marrow cells treated with a control compound is an indication of aactive and viable GM-CSF compound.

H. IFN-Gamma

It is an object of the present invention to encompass a method ofmodifying and/or remodeling IFN-gamma. IFN-gamma, otherwise known asType II interferon, in contrast to IFN alpha and IFN beta, is ahomodimeric glycoprotein comprising two subunits of about 21-24 kDa. Thesize variation is due to variable glycosylation patterns, usually notreplicated when reproduced recombinantly in various expression systemsknown in the art. IFN-gamma is a potent activator of macrophages,increases MHC class I molecule expression, and to a lesser extent, a MHCclass II molecule stimulatory agent. Further, IFN-gamma promotes T-celldifferentiation and isotype switching in B-cells. IFN-gamma is also welldocumented as a stimulator of neutrophils, NK cells, and antibodyresponses leading to phagocyte-mediated clearance. IFN-gamma has beenproposed as a treatment to be used in conjunction with infection byintracellular pathogens, such as tuberculosis and leishmania, and alsoas an anti-proliferative therapeutic, useful in conditions with abnormalcell proliferation as a hallmark, such as various cancers and otherneoplasias.

IFN-gamma has demonstrated potent immunological activity, but due tovariations in glycosylation from systems currently used to expressIFN-gamma, the potency, efficacy, biological half-life, and otherimportant factors of a therapeutic have been variable at best. Thepresent invention encompasses methods to correct this crucial defect.

A remodeled interferon-gamma peptide may be administered to a patientselected from the group consisting of a patient having chronicgranulomatous disease, a patient having malignant osteopetrosis, apatient having pulmonary fibrosis, a patient having tuberculosis, apatient having Cryptococcal meningitis, and a patient having pulmonaryMycobacterium avium complex (MAC) infection. Preferably, the patient isa human patient.

The nucleotide and amino acid sequences of IFN-gamma are presentedherein as SEQ ID NO:19 and SEQ ID NO:20, respectively (FIGS. 67A and67B, respectively). It will be readily understood that the sequences setforth herein are in no way limiting to the present invention. Incontrast, variants, derivatives, and mutants of IFN-gamma are well knownto the skilled artisan. As an example, U.S. Pat. No. 6,083,724 describesa recombinant avian IFN-gamma and U.S. Pat. No. 5,770,191 describesC-terminus variants of human IFN-gamma. In addition, U.S. Pat. No.4,758,656 describes novel IFN-gamma derivatives, and methods ofsynthesizing them in various expression systems. Therefore, the presentinvention is not limited to the sequences of IFN-gamma disclosedelsewhere herein, but encompasses all derivatives, variants, muteins,and the like well known in the art.

Expression systems for IFN-gamma are equally well known in the art, andinclude prokaryotic and eukaryotic systems, as well as plant and insectcell preparations, methods of which are known to the skilled artisan. Asan example, U.S. Pat. No. 4,758,656 describes a system for expressingIFN-gamma derivatives in E. coli, whereas U.S. Pat. No. 4,889,803describes an expression system employing Chinese hamster ovary cells andan SV40 promoter.

Assays for the biological activity of a remodeled IFN-gamma preparedaccording to the methods disclosed herein will be well known to one ofskill in the art. Biological assays for IFN-gamma expression can befound in, for example, U.S. Pat. No. 5,807,744. Briefly, IFN-gamma isadded to cultures of CD34⁺⁺CD38⁻ cells (100 cells per well) stimulatedby cytokine combinations to induce proliferation of CD34⁺⁺CD38⁻ cells,such as IL-3, c-kit ligand and either IL-1, IL-6 or G-CSF. Cellproliferation, and generation of secondary colony forming cells will beprofoundly inhibited in a dose dependent way, with near completeinhibition occurring at 5000 U/milliliter of IFN-gamma. As aconfirmatory test to the inhibitory effect of IFN-gamma, addition ofIFN-gamma antibodies can be performed as a control.

I. Alpha-Protease Inhibitor (α-Antitrypsin)

The present invention further includes a method for the remodeling ofalpha-protease inhibitor (A-1-PI, α-1-antitrypsin or α-1-trypsininhibitor), also known as alpha-antitrypsin. A-1-PI is a glycoproteinhaving molecular weight of 53 kDa. A-1-PI plays a role in controllingtissue destruction by endogenous serine proteases, and is the mostpronounced serine protease inhibitor in blood plasma. In particular,A-1-PI inhibits various elastases including neutrophil elastase.Elastase is a protease which breaks down tissues, and can beparticularly problematic when its activity is unregulated in lungtissue. This protease functions by breaking down foreign proteins.However, when API is not present in sufficient quantities to regulateelastase activity, the elastase breaks down lung tissue. In time, thisimbalance results in chronic lung tissue damage and emphysema. In fact,a genetic deficiency of A-1-PI has been shown to be associated withpremature development of pulmonary emphysema. A-1-PI replenishment hasbeen successfully used for treatment of this form of emphysema. Further,a deficiency of A-1-PI may also contribute to the aggravation of otherdiseases such as cystic fibrosis and arthritis, where leukocytes move into the lungs or joints to fight infection.

Therefore, A-1-PI could conceivably be used to treat diseases where animbalance between inhibitor and protease(s), especially neutrophilelastase, is causing progression of a disease state. Antiviral activityhas also been attributed to A-1-PI. In light of this, it logicallyfollows that the present invention is useful for the production ofA-1-PI that is safe, effective, and potent in the ever changingatmosphere of the lungs.

A remodeled A-1-P1peptide may be administered to a patient selected fromthe group consisting of a patient having congenital alpha-1-antitrypsindeficiency and emphysema, a patient having cystic fibrosis, and apatient having pulmonary fibrosis. Preferably, the patient is a humanpatient.

A-1-PI has been cloned and sequenced, and is set forth in SEQ ID NO:21and SEQ ID NO:22 (FIGS. 68A and 68B, respectively). As is understood byone of skill in the art, natural and engineered variants of A-1-PIexist, and are encompassed in the present invention. As an example, U.S.Pat. No. 5,723,316 describes A-1-PI derivatives having amino acidsubstitutions at positions 356-361 and further comprises an N-terminalextension of approximately three amino acids. U.S. Pat. No. 5,674,708describes A-1-PI analogs with amino acid substitutions at position 358in the primary amino acid sequence. The skilled artisan will readilyrealize that the present invention encompasses A-1-PI variants,derivatives, and mutants known or to be discovered.

Methods for the expression and determination of activity of a remodeledA-1-PI produced according to the methods of the present invention arewell known in the art, and are described in, for example, U.S. Pat. No.5,674,708 and U.S. Pat. No. 5,723,316. Briefly, biological activity canbe determined using assays for antichymotrypsin activity by measuringthe inhibition of the chymotrypsin-catalyzed hydrolysis of substrateN-suc-Ala-Ala-Pro-Phe-p-nitroanilide (0.1 ml of a 10 mM solution in 90%DMSO), as described in, for example, DelMar et al. (1979, Anal. Biochem.99:316). A typical chymotrypsin assay contains, in 1.0 milliliters: 100mM Tris-Cl buffer, pH 8.3, 0.005% (v/v) Triton X-100, bovine pancreaticchymotrypsin (18 kmmol) and A-1-PI of the present invention. The assaymixture is pre-incubated at room temperature for 5 minutes, substrate(0.01 ml of a 10 mM solution in 90% DMSO) is added and remainingchymotrypsin activity is determined by the rate of change in absorbanceat 410 mn caused by the release of p-nitroaniline. Measurements ofoptical absorbance are conducted at 25° C. using a spectrophotometerfitted with a temperature controlled sample compartment.

J. Glucocerebrosidase

The invention described herein further includes a method for themodification of glucocerebrosidase. Glucocerebrosidase is a lysosomalglycoprotein enzyme which catalyzes the hydrolysis of the glycolipidglucocerebroside to glucose and ceramide. Variants of glucocerebrosidaseare sold commercially as Cerezyme™ and Ceredase™, and is an approvedtherapeutic for the treatment of Gaucher disease. Ceredase™ is aplacental derived form of glucocerebrosidase with complete N-linkedstructures. Cerezyme™ is a recombinant variant of glucocerebrosidasewhich is 497 amino acids in length and is expressed in CHO cells. The 4N-linked glycans of Cerezyme have been modified to terminate in thetrimannose core.

Glucocerebrosidase is presently produced in recombinant mammalian cellcultures, and therefore reflects the glycosylation patterns of thosecells, usually rodent cells such as Chinese hamster ovary cells or babyhamster kidney cells, which differ drastically from those of humanglycosylation patterns, leading to, among other things, immunogenicityand lack of potency.

A remodeled glucocerebrosidase peptide may be administered to a patientselected from the group consisting of a patient having a lysosomalstorage disease, a patient having a glucocerebrosidase deficiency, and apatient having Gaucher disease. Preferably, the patient is a humanpatient.

The nucleic acid and amino acid sequences of glucocerebrosidase are setforth herein as SEQ ID NO:23 and 24 (FIGS. 69A and 69B, respectively).However, as will be appreciated by the skilled artisan, the sequencesrepresented herein are prototypical sequences, and do not limit theinvention. In fact, variants of glucocerebrosidase are well known, andare described in, for example, U.S. Pat. No. 6,015,703 describesenhanced production of glucocerebrosidase analogs and variants thereof.Further, U.S. Pat. No. 6,087,131 describes the cloning and sequencing ofyet another glucocerebrosidase variant. It is the intention of thepresent invention to encompass these and other derivatives, variants,and mutants known or to be discovered in the future.

Methods for the expression of glucocerebrosidase are well known in theart using standard techniques, and are described in detail in, forexample, U.S. Pat. No. 6,015,703. Assays for the biological efficacy ofa glucocerebrosidase molecule prepared according to the methods of thepresent invention are similarly well known in the art, and a mouseGaucher disease model for evaluation and use of a glucocerebrosidasetherapeutic is described in, for example, Marshall et al. (2002, Mol.Ther. 6:179).

K. TPA

The present invention further encompasses a method for the remodeling oftissue-type activator (TPA). TPA activates plasminogen to form plasminwhich dissolves fibrin, the main component of the protein substrate ofthe thrombus. TPA preparations were developed as a thrombolytic agentshaving a very high selectivity toward the thrombus in the thrombolytictreatment for thrombosis which causes myocardial infarction and cerebralinfarction.

Further, various modified TPA's have been produced by geneticengineering for the purpose of obtaining higher affinity to fibrin andlonger half-life in blood than that of natural TPA. TPA's are proteinsthat are generally extremely difficult to solubilize in water. Inparticular, the modified TPA's are more difficult to solubilize in waterthan, natural TPA, making very difficult the preparation of modifiedTPA's. Modified TPA's are thus difficult to dissolve in water at thetime of the administration to a patient. However, the modified TPA'shave various advantages, such as increased affinity for fibrin andlonger half-life in blood. It is the object of the present invention toincrease the solubility of modified TPA's.

A remodeled TPA peptide may be administered to a patient selected fromthe group consisting of a patient suffering from an acute myocardialinfarction and a patient suffering from an acute ischemic stroke. Aremodeled TPA peptide may also be administered to a patient to improveventricular function following an acute myocardial infarction, to reducethe incidence of congestive heart failure following an acute myocardialinfarction, or to reduce mortality associated with acute myocardialinfarction. A remodeled TPA peptide may also be administered to apatient to improve neurological recovery following an acute ischemicstroke or to reduce the incidence of disability or paralysis followingan acute ischemic stroke. Preferably, the patient is a human patient.

The nucleic and amino acid sequences of TPA are set forth herein as SEQID NO:25 and SEQ ID NO:26, respectively (FIGS. 70A and 70B,respectively). As described above, variants of TPA have been constructedand used in therapeutic applications. For example, U.S. Pat. No.5,770,425 described TPA variants in which some of all of the fibrindomain has been deleted. Further, U.S. Pat. No. 5,736,134 describes TPAin which modifications to the amino acid at position 276 are disclosed.The skilled artisan, when equipped with the present disclosure and theteachings herein, will readily realize that the present inventioncomprises the TPA sequences set forth herein, as well as those variantswell known to one versed in the literature.

The expression of TPA from a nucleic acid sequence encoding the same iswell known in the art, and is described, in detail, in, for example,U.S. Pat. No. 5,753,486. Assays for determining the biologicalproperties of a TPA molecule prepared according to the methods of thepresent invention are similarly well known in the art. Briefly, a TPAmolecule synthesized as disclosed elsewhere herein can be assayed fortheir ability to lyse fibrin in the presence of saturatingconcentrations of plasminogen, according to the method of Carlsen et al.(1988, Anal. Biochem. 168: 428). The in vitro clot lysis assay measuresthe activity of tissue-type activators by turbidimetry using amicrocentrifugal analyzer. A mixture of thrombin and TPA is centrifugedinto a mixture of fibrinogen and plasminogen to initiate clot formationand subsequent clot dissolution. The resultant profile of absorbanceversus time is analyzed to determine the assay endpoint. Activities ofthe TPA variants are compared to a standard curve of TPA. The bufferused throughout the assay is 0.06M sodium phosphate, pH 7.4 containing0.01% (v/v) TWEEN 80 and 0.01% (w/v) sodium azide. Human thrombin is ata concentration of about 33 units/ml. Fibrinogen (at 2.0 mg/ml clottableprotein) is chilled on wet ice to precipitate fibronectin and thengravity filtered. Glu-plasminogen is at a concentration of 1 mg/ml. Theanalyzer chamber temperature is set at 37° C. The loader is set todispense 20 microliters of TPA (about 500 nanograms/milliliter to about1.5 micrograms per milliliter) as the sample for the standard curve, or20 microliters of variant TPAs at a concentration to cause lysis withinthe range of the standard curve. Twenty microliters of thrombin as thesecondary reagent, and 200 microliters of a 50:1 (v/v) fibrinogen:plasminogen mixture as the primary reagent. The absorbance/time programis used with a 5 min incubation time, 340-nanometer-filter and 90 secondinterval readings.

L. IL-2

The present invention further encompasses a method for the remodelingand modification of IL-2. IL-2 is the main growth factor of Tlymphocytes and increases the humoral and cellular immune responses bystimulating cytotoxic CD8 T cells and NK cells. IL-2 is thereforecrucial in the defense mechanisms against tumors and viral infections.IL-2 is also used in therapy against metastatic melanoma and renaladenocarcinoma, and has been used in clinical trials in many forms ofcancer. Further, IL-2 has also been used in HIV infected patients whereit leads to a significant increase in CD4 counts.

Given the success IL-2 has demonstrated in the management and treatmentof life-threatening diseases such as various cancers and AIDS, itfollows that the methods of the present invention would be useful fordeveloping an IL-2 molecule that has a longer biological half-life,increased potency, and in general, a therapeutic profile more similar towild-type IL-2 as it is synthesized secreted in the healthy human.

A remodeled IL-2 peptide may be administered to a patient selected fromthe group consisting of a patient having metastatic renal cellcarcinoma, a patient having metastatic melanoma, a patient havingovarian cancer, a patient having Acute Myelogenous Leukemia (AML), apatient having non-Hodgkin's lymphoma (NHL), a patient infected withHIV, and a patient infected with Hepatitis C. A remodeled IL-2 peptidemay also be useful for administeration to a patient as a cancer vaccineadjuvant. Preferably, the patient is a human patient.

IL-2 has been cloned and sequenced, and the nucleic acid and amino acidsequences are presented herein as SEQ ID NO:27 and SEQ ID NO:28 (FIGS.71A and 71B, respectively). The present invention should in no way beconstrued as limited to the IL-2 nucleic acid and amino acid sequencesset forth herein. Variants of IL-2 are described in, for example, U.S.Pat. No. 6,348,193, in which the asparagine at position 88 issubstituted for arginine, and in U.S. Pat. No. 5,206,344, in which apolymer comprising IL-2 variants with various amino acid substitutionsis described. The present invention encompasses these IL-2 variants andothers well known in the art.

Methods for the expression and to determine the activity of IL-2 arewell known in the art, and are described in, for example, U.S. Pat. No.5,417,970. Briefly, expression of IL-2, or variants thereof, can beaccomplished in a variety of both prokaryotic and eukaryotic systems,including E. Coli, CHO cells, BHK cells, insect cells using abaculovirus expression system, all of which are well known in the art.

Assays for the activity of a modified IL-2 prepared according to themethods of the present invention can proceed as follows. Peripheralblood lymphocytes can be separated from the erythrocytes andgranulocytes by centrifuging on a Ficoll-Hypaque (Pharmacia, Piscataway,N.J.) gradient by the method described in, for example, A. Boyum et al.(Methods in Enzymology, 1984, Vol. 108, page 88, Academic Press, Inc.).Lymphocytes are subsequently washed about three times in culture mediumconsisted of RPMI 1640 (Gibco-BRL, La Jolla, Calif.) plus 10% AB humanserum (CTS Purpan, Toulouse, France) inactivated by heat (1 hour at 56°C.), 2 mM sodium pyruvate, 5 mM HEPES, 4 mM L-glutamine, 100 U/mlpenicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B(complete medium). Adhesive cells (monocytes and macrophages) areeliminated by adhesion to plastic and the remainder of the cells aresuspended in complete medium at a concentration of about 5 to 10×10⁵cells per milliliter and seeded in culture flasks at a density of about1-2×10⁵ cells per square centimeter. Flasks are then incubated at 37° C.in a 5% CO₂ atmosphere for about 1 hour, after which the non-adhesivelymphocytes are recovered by aspiration after gentle agitation of theculture flasks.

Non-adhesive lymphocytes are washed once and cultivated at aconcentration of about 10⁵ cells per milliliter in complete medium inthe presence of the IL-2 of the present invention for about 48 hours inan incubator as described above. The cells are then washed once.

The cytotoxic activity of the cells is evaluated after about 4 hours ofcontact with target cells of the human T lymphoid line C8166-45/C63 (HT1cells) resistant to NK cell cytotoxicity, as described by Salahuddin etal. (1983, Virology 129: 51-64; 1984, Science: 223, 703-707). 6×10⁵ HT1cells are radio-tagged with about 200 μCi of ⁵¹Cr (sodium chromate,Amersham, Arlington Heights, Ill.) at 37° C. for about 1 hour incomplete medium without serum, and then washed several times. The targetcells and effective cells are distributed in round-bottomedmicrotitration plates with varying ratios of effective cells to targetcells (50:1, 10:1, 1:1). The microtitration plates are centrifuged and,after incubation as described above, the supernatant from each well isrecovered and the radioactivity is measured using a gamma counter.Cytotoxicity is determined from the quantity of ⁵¹Cr released by deadtarget cells. Non-specific cytotoxicity is determined from the amount ofradioactivity spontaneously released from the target cells in theabsence of effective cells.

The present method is just one of many well known in the art formeasuring the cytotoxicity of effector cells, and is should not beconstrued as limiting to the present invention.

M. Factor VIII

The invention further encompasses a method for the remodeling andmodification of Factor VIII. As described earlier for Factor VII andFactor IX, Factor VIII is a critical component of the blood coagulationpathway. Human Factor VIII, (antihemophilic factor; FVIII:C) is a humanplasma protein consisting of 2 peptides (light chain molecular weight of80 kDa and heavy chain molecular weight variable from 90 to 220 kDa,depending on glycosylation state). It is an essential cofactor in thecoagulation pathway and is required for the conversion of Factor X intoits active form (Factor Xa). Factor VIII circulates in plasma as anon-covalent complex with von Willibrand Factor (aka FVIII:RP), a dimerof a 2050 aa peptide (See, U.S. Pat. No. 6,307,032). Bloodconcentrations of Factor VIII below 20% of normal cause a bleedingdisorder designated hemophilia A. Factor VIII blood levels less than 1%result in a severe bleeding disorder, with spontaneous joint bleedingbeing the most common symptom.

Similar to other blood coagulation factors, Factor VIII is a therapeuticwith a great deal of potential for the treatment of various bleedingdisorders, such as hemophilia A and hemophilia B. Due to theglycosylation of the heavy chain, current methods for the preparation ofFactor VIII from recombinant cells results in a product that is not aseffective as natural Factor VIII. Purification methods from human plasmaresult in a crude composition that is less effective and more difficultto prepare than recombinant Factor VIII. The current invention seeks toimprove this situation.

A remodeled Factor VIII peptide may be administered to a patientselected from the group consisting of a patient having von Willebrand'sdisease, a patient having Hemophilia A, a patient having Factor VIII:Cdeficiency, a patient having fibrinogen deficiency, a patient havingFactor XIII deficiency, and a patient having acquired Factor VIIIinhibitors (acquired hemophilia). A remodeled Factor VIII peptide mayalso be administered to a patient to prevent, treat or control bleedingor hemorrhagic episodes. Preferably, the patient is a human patient.

The nucleic acid and amino acid sequences of Factor VIII are presentedherein as SEQ ID NO:29 and SEQ ID NO:30, respectively (FIGS. 72A and72B, respectively). The art is rife with variants of Factor VIII, asdescribed in, for example, U.S. Pat. No. 5,668,108, in which theaspartic acid at position 1241 is replaced by a glutamic acid with theaccompanying nucleic acid changes as well. U.S. Pat. No. 5,149,637describes a Factor VIII variants comprising the C-terminal fraction,either glycosylated or unglycosylated, and U.S. Pat. No. 5,661,008describes a Factor VIII variant comprising amino acids 1-740 linked toamino acids 1649 to 2332 by at least 3 amino acid residues. Therefore,variants, derivatives, modifications and complexes of Factor VIII arewell known in the art, and are encompassed in the present invention.

Expression systems for the production of Factor VIII are well known inthe art, and include prokaryotic and eukaryotic cells, as exemplified inU.S. Pat. Nos. 5,633,150, 5,804,420, and 5,422,250.

To determine the biological activity of a Factor VIII moleculesynthesized according the methods of the present invention, the skilledartisan will recognize that the assays described herein for theevaluation of Factor VII and Factor IX are applicable to Factor VIII.

N. Urokinase

The present invention also includes a method for the remodeling and/ormodification of urokinase. Urokinase is a serine protease whichactivates plasminogen to plasmin. The protein is synthesized in avariety of tissues including endothelium and kidney, and is excreted intrace amounts into urine. Purified urokinase exists in two active forms,a high molecular weight form (HUK; approximately 50 kDa) and a lowmolecular weight form (LUK; approximately 30 kDa). LUK has been shown tobe derived from HUK by a proteolysis after lysine 135, releasing thefirst 135 amino acids from HUK. Conventional wisdom has held that HUK orLUK must be converted to proteolytically active forms by the proteolytichydrolysis of a single chain precursor, also termed prourokinase,between lysine 158 and isoleucine 159 to generate a two-chain activatedform (which continues to correspond to either HUK or LUK). Theproteolytically active urokinase species resulting from this hydrolyticclip contains two amino acid chains held together by a single disulfidebond. The two chains formed by the activation clip are termed the A orA₁ chains (HUK or LUK, respectively), and the B chain comprising theprotease domain of the molecule.

Urokinase has been shown to be an effective thrombolytic agent. However,since it is produced naturally in trace quantities the cost of theenzyme is high for an effective dosage. Urokinase has been produced inrecombinant cell culture, and DNA encoding urokinase is known togetherwith suitable vectors and host microorganisms. Present compositionscomprising urokinase and methods for producing urokinase recombinantlyare hampered by a product that has deficient glycosylation patterns, andgiven the complex proteolytic cleavage events surrounding the activationof urokinase, this aberrant glycosylation leads to a less effective andless potent product.

A remodeled urokinase peptide may be administered to a patient selectedfrom the group consisting of a patient having an embolism, a patienthaving an acute massive pulmonary embolism, and a patient havingcoronary artery thrombosis. Preferably, the patient is a human patient.A remodeled urokinase peptide may also be used to restore patency to anintravenous catheter, including a central venous catheter obstructed byclotted blood or fibrin.

The sequence of the nucleotides encoding the primary amino acid chain ofurokinase are depicted in SEQ ID NO:33 and SEQ ID NO:34 (FIGS. 73A and73B, respectively). Variants of urokinase are well known in the art, andtherefore the present invention is not limited to the sequences setforth herein. In fact, the skilled artisan will readily realize thaturokinase variants described in, for example U.S. Pat. Nos. 5,219,569,5,648,253, and 4,892,826, exist as functional moieties, and aretherefore encompassed in the present invention.

The expression and evaluation of a urokinase molecule prepared accordingto the methods of the present invention are similarly well known in theart. As a non-limiting example, the expression of urokinase in varioussystems is detailed in U.S. Pat. No. 5,219,569. An assay for determiningthe activity and functionality of a urokinase prepared in accordance tothe methods set forth herein are described throughout the literature,and are similar to assays for other plasminogen and fibrin relatedassays described elsewhere throughout. One example of an assay todetermine the activity of an urokinase molecule synthesized as describedherein can be as described in, for example, Ploug, et al. (1957,Biochim. Biophys. Acta 24: 278-282), using fibrin plates comprising1.25% agarose, 4.1 mg/ml human fibrinogen, 0.3 units/ml of thrombin and0.5 μg/ml of soybean trypsin inhibitor.

O. Human DNase

The present invention further encompasses a method for the remodelingand/or modification of recombinant human DNase. Human DNase I has beentested as a therapeutic agent and was shown to diminish the viscosity ofcystic fibrosis mucus in vitro. It has been determined that purulentmucus contains about 10-13 mg/ml of DNA, an ionic polymer predicted toaffect the rheologic properties of airway fluids. Accordingly, bovinepancreatic DNase I, an enzyme that degrades DNA, was tested as amucolytic agent many years ago but did not enter clinical practice,because of side effects induced by antigenicity and/or contaminatingproteases. Recombinant human DNase is currently used as a therapeuticagent to alleviate the symptoms of diseases such as cystic fibrosis.

A remodeled rDNase peptide may be administered to a patient havingcystic fibrosis. A remodeled rDNase peptide may also be administered toa cystic fibrosis patient to improve pulmonary function. Preferably, thepatient is a human patient.

Similar to DNase derived from bovine sources, recombinant human DNaseposes some problems, mostly due to lowered efficacy due to improperglycosylation imparted by mammalian expression systems currently in use.The present invention describes a method for remodeling DNase, leadingto increased efficacy and better therapeutic results.

The nucleotide and amino acid sequences of human DNAse are presentedherein as SEQ ID NO:39 and SEQ ID NO:40 (FIGS. 74A and 74B,respectively). Variants of the peptide comprising DNase are well knownin the art. As an example, U.S. Pat. No. 6,348,343 describes a humanDNase with multiple amino acid substitutions throughout the primarystructure. Additionally, U.S. Pat. No. 6,391,607 describes a hyperactivevariant of DNase with multiple amino acid substitutions at positions 9,14, 74, 75, and 205. The present examples, and others well known in theart or to be discovered in the future are encompassed in the presentinvention.

Expression systems for producing a DNase peptide are well known to theskilled artisan, and have been described in prokaryotic and eukaryoticsystems. For example, PCT Patent Publication No. WO 90/07572 describesthese methods in considerable detail.

Assays to determine the biological activity of a DNase moleculedeveloped according to the methods of the present invention are wellknown in the art. As an example, but in no way meant to be limiting tothe present invention, an assay to determine the DNA-hydrolytic activityof human DNase I is presented herein. Briefly, two different plasmiddigestion assays are used. The first assay (“supercoiled DNA digestionassay”) measures the conversion of supercoiled double-stranded plasmidDNA to relaxed (nicked), linear, and degraded forms. The second assay(“linear DNA digestion assay”) measured the conversion of lineardouble-stranded plasmid DNA to degraded forms. Specifically, DNaseprepared according to the methods of the present invention is added to160 microliters of a solution comprising 25 micrograms per milliliter ofeither supercoiled plasmid DNA or EcoRI-digested linearized plasmid DNAin 25 mM HEPES, pH 7.1, 100 μg/ml bovine serum albumin, 1 mM MgCl₂, 2.5mM CaCl₂, 150 mM NaCl, and the samples are incubated at roomtemperature. At various times, aliquots of the reaction mixtures areremoved and quenched by the addition of 25 mM EDTA, together with xylenecyanol, bromophenol blue, and glycerol. The integrity of the plasmid DNAin the quenched samples is analyzed by electrophoresis of the samples onagarose gels. After electrophoresis, the gels are stained with asolution of ethidium bromide and the DNA in the gels is visualized byultraviolet light. The relative amounts of supercoiled, relaxed, andlinear forms of plasmid DNA are determined by scanning the gels with afluorescent imager (such as the Molecular Dynamics Model 575FluorImager) and quantitating the amount of DNA in the bands of the gelthat correspond to the different forms.

P. Insulin

The invention further includes a method for remodeling insulin. Insulinis well known as the most effective treatment for type I diabetes, inwhich the beta islet cells of the pancreas do not produce insulin forthe regulation of blood glucose levels. The ramifications of diabetesand uncontrolled blood glucose include circulatory and foot problems,and blindness, not to mention a variety of other complications thateither result from or are exacerbated by diabetes.

Prior to the cloning and sequencing of human insulin, porcine insulinwas used as a treatment for diabetes. Insulin is now producedrecombinantly, but the short, 51 amino acid sequence of the maturemolecule is a complex structure comprising multiple sulfide bonds.Current methods to recombinantly produce insulin result in a productthat lacks similarity to the native protein as produced in healthynon-diabetic subjects. The present invention seeks to repair this flaw.

A remodeled insulin peptide may be administered to a patient selectedfrom the group consisting of a patient having Type I Diabetes (diabetesmellitus) and a patient having Type 2 diabetes mellitus who requiresbasal (long-acting) insulin for the control of hyperglycemia. Aremodeled insulin peptide may also be administered to a diabetic patientto control hyperglycemia. Preferably, the patient is a human patient.

The nucleotide and amino acid sequence of human insulin is portrayed inSEQ ID NO:43 and SEQ ID NO:44, respectively (FIGS. 75A and 75B,respectively). Variants of insulin are abundant throughout the art. U.S.Pat. No. 6,337,194 describes insulin fusion protein analogs, U.S. Pat.No. 6,323,311 describes insulin derivatives comprising a cyclicanhydride of a dicarboxylic acid, and U.S. Pat. No. 6,251,856 describesan insulin derivative comprising multiple amino acid substitutions and alipophilic group. The skilled artisan will recognize that the followingexamples of insulin derivatives are in no way exhaustive, but simplyrepresent a small sample of those well known in the art. Therefore, thepresent invention comprises insulin derivatives known or to bediscovered.

Expression systems for the production of insulin are well known in theart, and can be accomplished using molecular biology techniques asdescribed in, for example, Sambrook et al. (1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York).

Assays to determine the functionality of an insulin molecule preparedaccording to the methods of the present invention are similarly wellknown in the art. For example, an in vivo model of glucose depressioncan be used to evaluate the biological activity of insulin synthesizedusing the methods of the present invention. Useful for this purpose is arat model. The animals are fasted overnight (16 hours) prior to theexperiment, and then anesthetized with intraperitoneally administeredsodium pentobarbital or another suitable anesthetic such as ketamine.Each animal receives an i.v. injection (tail vein) of the particularinsulin derivative (20 μg/ml/kg). Blood samples are taken from thejugular vein 15 and 5 minutes before injection and 15, 30, 60, 90, 120,180, and 240 minutes after injection. Blood glucose levels are measuredwith a blood glucose monitor, available from a variety of commercialsuppliers.

Q. Hepatitis B Vaccines (HBsAg)

The present invention further comprises a method for the remodeling theantigen used in hepatitis B vaccines (HbsAg or Hepatitis B sAg). HBsAgis a recombinantly produced surface antigen of the hepatitis BS-protein, and is used to illicit an immune response to the hepatitis Bvirus, an increasing dangerous virus that results in, among otherthings, liver disease including cirrhosis and carcinoma, and results inover 1 million deaths worldwide annually. Currently the HBsAg vaccine isadministered three times over a six month interval to illicit aprotective and neutralizing immune response.

HBsAg is currently produced in yeast strains, and therefore reflects theglycosylation patterns native to a fungus. The present inventionprovides a method to remodel HBsAg, resulting in among other things,improved immunogenicity, antibodies with improved affinity for thevirus, and the like.

A remodeled HBsAg peptide may be administered to a patient to immunizethe patient against disease caused by a Hepatitis B virus. A remodeledHBsAg peptide may also be administered to a predialysis patient or adialysis patient to immunize the patient against disease caused by aHepatitis B virus. Preferably, the patient is a human patient.

The sequences of the S-protein from a Hepatitis B virus (HBsAg) nucleicacid and primary amino acid chain are set forth herein as SEQ ID NO:45and SEQ ID NO:46 (FIGS. 76A and 76B, respectively). The nucleotide is1203 bases in length. The amino acid is 400 residues long. The last 226amino acid residues are the small S-antigen, which is used in theGlaxoSmithKline vaccine and the Merck vaccine. Fifty-five amino acidsupstream from the small S-antigen is the Pre-S start codon. The Pre-S+Sregions are the middle S antigen, which is used in the Aventis Pasteurvaccine. From the first start codon to the Pre-S start codon comprisesthe rest of the S-protein, and is called the large S-protein. This isbut one example of the HBsAg used in vaccines, and other subtypes arewell known, as exemplified in GenBank Acc Nos.: AF415222, AF415221,AF415220, and AF415219. The sequences presented herein are simplyexamples of HBsAg known in the art. Similar antigens have been isolatedfrom other strains of hepatitis B virus, and may or may not have beenevaluated for antigenicity and potential as vaccine candidates. Thepresent invention therefore encompasses hepatitis B vaccine S-proteinsurface antigens known or to be discovered.

Expression of an HBsAg in an expression system is a routine procedurefor one of skill in the art, and is described in, for example, U.S. Pat.No. 5,851,823. Assays for the immunogenicity of a vaccine are well knownin the art, and comprise various tests for the production ofneutralizing antibodies, and employ techniques such as ELISA,neutralization assays, Western blots, immunoprecipitation, and the like.Briefly, a sandwich ELISA for the detection of effective anti-HBsAgantibodies is described. The Enzygnost HBsAg assay (Aventis Behring,King of Prussia, Pa.) is used for such methods. Wells are coated withanti-HBs. Serum plasma or purified protein and appropriate controls areadded to the wells and incubated. After washing, peroxidase-labeledantibodies to HBsAg are reacted with the remaining antigenicdeterminants. The unbound enzyme-linked antibodies are removed bywashing and the enzyme activity on the solid phase is determined bymethods well known in the art. The enzymatically catalyzed reaction ofhydrogen peroxide and chromogen is stopped by adding diluted sulfuricacid. The color intensity is proportional to the HBsAg concentration ofthe sample and is obtained by photometric comparison of the colorintensity of the unknown samples with the color intensities of theaccompanying negative and positive control sera.

R. Human Growth Hormone

The present invention further encompasses a method for the remodeling ofhuman growth hormone (HGH). The isoform of HGH which is secreted in thehuman pituitary, consists of 191 amino acids and has a molecular weightof about 21,500. The isoform of HGH which is made in the placenta is aglycosylated form. HGH participates in much of the regulation of normalhuman growth and development, including linear growth (somatogenesis),lactation, activation of macrophages, and insulin-like and diabetogeniceffects, among others.

HGH is a complex hormone, and its effects are varied as a result ofinteractions with various cellular receptors. While compositionscomprising HGH have been used in the clinical setting, especially totreat dwarfism, the efficacy is limited by the absence of glycosylationof the HGH produced recombinantly.

A remodeled HGH peptide may be administered to a patient selected fromthe group consisting of a patient having a growth hormone deficiency, apatient having Turner syndrome, a patient having growth failure due to alack of adequate endogenouse growth hormone secretion, a patient havinggrowth failure due to Prader-Willi syndrome (PWS), a patient havinggrowth failure associated with chronic renal insufficiency, and apatient having AIDS associated wasting or cachexia. A remodeled HGHpeptide may also be administered to a patient having short stature.Preferably, the patient is a human patient.

The nucleic and amino acid sequence of HGH are set forth elsewhereherein as SEQ ID NO:47 and SEQ ID NO:48 (FIGS. 77A and 77B,respectively). The skilled artisan will recognize that variants,derivatives, and mutants of HGH are well known. Examples can be found inU.S. Pat. No. 6,143,523 where amino acid residues at positions 10, 14,18, 21, 167, 171, 174, 176 and 179 are substituted, and in U.S. Pat. No.5,962,411 describes splice variants of HGH. The present inventionencompasses these HGH variants known in the art of to be discovered.

Methods for the expression of HGH in recombinant cells is described in,for example, U.S. Pat. No. 5,795,745. Methods for expression of HGH in,inter alia, prokaryotes, eukaryotes, insect cell systems, plants, and invitro translation systems are well known in the art.

An HGH molecule produced using the methods of the current invention canbe assayed for activity using a variety of methods known to the skilledartisan. For example, U.S. Pat. No. 5,734,024 describes a method todetermine the biological functionality of an expressed HGH.

S. Anti-Thrombin III

Antithrombin (antithrombin III, AT-III) is a potent inhibitor of thecoagulation cascade in blood. It is a non-vitamin K-dependent proteasethat inhibits the action of thrombin as well as other procoagulantfactors (e.g., Factor Xa). Congenital antithrombin III deficiency is anautosomal dominant disorder in which an individual inherits one copy ofa defective gene. This condition leads to increased risk of venous andarterial thrombosis, with onset of clinical manifestations typicallypresenting in young adulthood. Severe congenital antithrombin IIIdeficiency, in which the individual inherits two defective genes, is anautosomal recessive condition associated with increased thrombogenesis,typically noted in infancy. Acquired antithrombin III deficiency mostcommonly is seen in situations where there is inappropriate activationof the coagulation system. Common conditions that result in acquiredantithrombin III deficiency include disseminated intravascularcoagulation, microangiopathic hemolytic anemias due to endothelialdamage (i.e., Hemolytic-uremic syndrome), and veno-occlusive disease(VOD) seen in patients undergoing bone marrow transplant. AT-IIIdeficiency may be corrected acutely by infusions of AT-III concentrates.

A remodeled AT-III peptide may be administered to a patient selectedfrom the group consisting of a patient having a hereditary AT-IIIdeficiency in connection with a surgical or obstetrical procedure and ahereditary AT-III deficient patient having a thromboembolism.Preferably, the patient is a human patient.

Antithrombin III (AT-III) is an α2-glycoprotein of molecular weight58,000. It is sold commercially as Thrombate III™ (Bayer Corp., WestHaven, Conn.). The nucleic acid and amino acid sequences of humanantithrombin III are displayed in FIGS. 78A (SEQ ID NO:63) and 78B (SEQID NO:64), respectively.

Methods to make anti-thrombin III are well know to those in the art. Forexample, published nucleic acid and amino acid sequences are availablefor human antithrombin III (see, U.S. Pat. No. 4,517,294) and mutants ofhuman antithrombin III (see, U.S. Pat. Nos. 5,420,252, 5,618,713,5,700,663). The methods of the invention may be used with any of theseamino acid sequences and any nucleic acid sequences that encode them,but are not limited to these sequences. Exemplary methods to producerecombinant antithrombin III are well known in the art, and several aredescribed in U.S. Pat. Nos. 5,420,252, 5,843,705, 6,441,145 and5,994,628. Exemplary methods to purify recombinant antithrombin III aredescribed in U.S. Pat. Nos. 5,989,593, 6,268,487, 6,395,888, 6,395,881,6,451,978 and 6,518,406.

There are many known uses for recombinant antithrombin III. AntithrombinIII can be used as a anticoagulant during surgery (U.S. Pat. Nos.5,252,557, 5,182,259), as part of a pharmaceutical preparation or methodto inhibit thrombosis (U.S. Pat. Nos. 5,565,471, 6,001,820), and toreduce the adverse side effects of cellular transplantation (U.S. Pat.No. 6,387,366). Additionally, antithrombin III preparations can be usedto increase placental blood flow (U.S. Pat. No. 5,888,964), inhibitfertilization (U.S. Pat. No. 5,545,615), treat asthma (U.S. Pat. No.6,355,626) and treat arthritis (U.S. Pat. No. 5,252,557) and otherinflammatory processes (U.S. Pat. No. 6,399,572). Antithrombin III canalso be used to manufacture replacement blood plasma (U.S. Pat. Nos.4,900,720) or prepare a stabilized cellular blood product (U.S. Pat. No.6,139,878) for transfusions. Antithrombin III may be administered as apharmaceutical preparation (U.S. Pat. Nos. 5,084,273, 5,866,122,6,399,572, 6,156,731 and 6,514,940) or using gene therapy methodology(U.S. Pat. No. 6,410,015). Compositions comprising antithrombin m can beused as tissue adhesives (U.S. Pat. No. 6,500,427) or lubricants formedical devices that are introduced to the patient (U.S. Pat. No.6,391,832). Antithrombin III can also be used to coat endovascularstents (U.S. Pat. Nos. 6,355,055, 6,240,616, 5,985,307, 5,685,847 and5,222,971), ocular implants (U.S. Pat. No. 5,944,753) and prostheses ingeneral (U.S. Pat. Nos. 6,503,556, 6,491,965 and 6,451,373).Antithrombin III can also be used in methods to locate an internalbleeding site in a patient (U.S. Pat. No. 6,314,314) and to determinehemostatic dysfunction in a patient (U.S. Pat. No. 6,429,017).

T. Human Chorionic Gonadotropin

Human Chorionic Gonadotropin (hCG) is a glycoprotein composed of analpha subunit and a beta subunit. HCG is closely related to two othergonadotropins, luteinizing hormone (LH) and follicle stimulating hormone(FSH), as well as thyroid stimulating hormone (TSH), all three of whichare glycoprotein hormones. The alpha subunits of these variousglycoprotein hormones are structurally very similar, but the betasubunits differ in amino acid sequence.

The nucleic acid and amino acid sequences of the human chorionicgonadotropin α-subunit are displayed in FIGS. 79A (SEQ ID NO:69) and 79B(SEQ ID NO:70), respectively. The nucleic acid and amino acid sequencesof the human chorionic gonadotropin β-subunit are displayed in FIGS. 79C(SEQ ID NO:71) and 79D (SEQ ID NO:72), respectively.

Human chorionic gonadotropin is used in an infertility treatment topromote ovulation or release of an egg from the ovary in women who donot ovulate on their own. Human chorionic gonadotropin is also given toyoung males to treat undescended or underdeveloped testicles. It is usedin men to stimulate the production of testosterone. Some physicians alsoprescribe human chorionic gonadotropin for men having erictiledysfunctionor lack of sexual desire, and for treatment of male“menopause.”

A remodeled hCG peptide may be administered to a patient selected fromthe group consisting of a patient undergoing assisted reproductivetechnology (ART), a patient undergoing in vitro fertilization (IVF), apatient undergoing embryo transfer, an infertile patient, a male patienthaving prepubertal cryptoorchidism not due to anatomical obstruction,and a male patient having hypogonadotropic hypogonadism. A remodeled hCGpeptide may also be administered to induce final follicular maturationand early luteinization in an infertile female patient, wherein theinfertile female patient has undergone pituitary desensitization andpretreatment with follicle stimulating hormones. A remodeled hCG peptidemay also be administered to induce ovulation and pregnancy in ananovulatory infertile patient. Preferably, the patient is a humanpatient.

Methods to make human chorionic gonadotropin are well known in the art.The heterodimeric hCG can be recombinantly made in any one of manyexpression systems currently used for industrial manufacture ofrecombinant proteins. One method of making recombinant hCG is describedin U.S. Pat. No. 5,639,639. Methods for making recombinant heterodimericproteins by expressing both subunits in the same cell are, in general,well known in the art, and several methods are described in the U.S.Pat. No. 5,643,745 (expression in a filamentous fungus), U.S. Pat. Nos.5,985,611 and 6,087,129 (expression in secretory cells). Alternatively,each subunit can be expressed individually in cells, and the twosubunits later brought together in vitro for assembly into theheterdimer.

Methods for using human chorionic gonadotropin are numerous and wellknown in the art. Commonly, hCG is used to induce or synchronizeovulation in mammals (see, U.S. Pat. Nos. 6,489,288, 5,589,457,5,532,155, 4,196,123, 4,062,942 and 4,845,077). Additionally, hCG can beused in pregnancy tests, and in particular agglutination-based tests(see, U.S. Pat. Nos. 3,991,175, 4,003,988, 4,071,314 and 4,088,749). hCGcan also be used in a contraceptive vaccine (see, U.S. Pat. Nos.4,161,519 and 4,966,888). In addition, hCG can be used to treatconditions related to aging and altered hormonal balance such as benignprostatic hypertrophy (see, U.S. Pat. No. 5,610,136) and central nervoussystem diseases common in the elderly (see, U.S. Pat. No. 4,791,099).

Alternatively, hCG can be used to detect and treat cancers that expresshCG or one of its subunits. hCG-expressing tumors include, but are notlimited to, breast, prostate, ovary and stomach carcinomas, andneuroblastomas such as Karposi's sarcoma. Antibodies can be raised tohCG which has been glycoremodeled so as to have glycan structuressimilar to those found on the tumor-expressed hCG, and these antibodiesmay be used to detect hCG-expressing tumors in patients according tomethods well known in the art (see, U.S. Pat. Nos. 4,311,688, 4,478,815and 4,323,546). Additionally, remodeled hCG can be used to raise animmune response to a tumor that is expressing hCG (see, U.S. Pat. Nos.5,677,275, 5,762,931, 5,877,148, 4,970,071 and 4,966,753).

hCG can also be used in methods to generally immunomodulate an animal,such as described in U.S. Pat. Nos. 5,554,595, 5,851,997 and 5,700,781.In addition, hCG can be used as an inhibitor of the matrixmetalloprotease in conditions benefiting from such treatment, such aschronic inflammatory diseases, multiple sclerosis andangiogenesis-dependent diseases (see, U.S. Pat. No. 6,444,639).

U. α-Iduronidase

α-Iduronidase is sold commercially as Aldurazyme™ (BioMarin andGenzyme). It is useful for replacement therapy for the treatment of MPSI, a lysosomal storage disease. MPS I (also known as Hurler disease) isa genetic disease caused by the deficiency of alpha-L-iduronidase, anenzyme normally required for the breakdown of certain complexcarbohydrates known as glycosaminoglycans (GAGs). The normal breakdownof GAGs is incomplete or blocked if the enzyme is not present insufficient quantity. The cell is then unable to excrete the carbohydrateresidues and they accumulate in the lysosomes of the cell and cause MPSI.

A remodeled alpha-iduronidase peptide may be administered to a patientselected from the group consisting of a patient having a lysosomalstorage disease, a patient having an alpha-L-iduronidase deficiency, apatient having mucopolysaccaridosis I (MPS I), and a patient havingHurler disease. Preferably, the patient is a human patient.

Methods to produce and purify α-iduronidase, as well as methods to treatcertain genetic disorders including α-L-iduronidase deficiency andmucopolysaccharidosis I (MPS 1) are described in U.S. Pat. No.6,426,208. The nucleic acid and amino acid sequences of humanα-iduronidase are found in FIGS. 80A (SEQ ID NO:65) and 80B (SEQ IDNO:66), respectively.

V. α-Galactosidase A

α-Galactosidase A (also known as agalsidase beta) is sold commerciallyas Fabrazyme™ (Genzyme). α-Galactosidase A is useful for the treatmentof Fabry disease. Fabry disease is a rare, inherited disorder caused bythe deficiency of the essential enzyme α-galactosidase. Without thisenzyme, Fabry patients are unable to breakdown a fatty acid substance intheir body called globotriasylceramide (GL-3), which accumulates incells in the blood vessels of the heart, kidney, brain and other vitalorgans. The progressive buildup of this substance puts patients a riskfor stroke, heart attack, kidney damage and debilitating pain. Mostpatients develop kidney failure during adulthood, and severe organcomplications lead to death around age forty.

A remodeled alpha-galactosidase A peptide may be administered to apatient selected from the group consisting of a patient having alysosomal storage disease, a patient having an alpha-galactosidase Adeficiency, and a patient having Fabry disease. Preferably, the patientis a human patient.

The α-galactosidase A enzyme is a lysosomal enzyme which hydrolyzesglobotriaosylceramide and related glycolipids which have terminalα-galactosidase linkages. It is a 45 kDa N-glycosylated protein encodedon the long arm of the X chromosome. The initial glycosylated forms(Mr=55,000 to 58,000) synthesized in human fibroblasts or Chang livercells are processed to a mature glycosylated form (Mr=50,000). Themature active enzyme as purified from human tissues and plasma is ahomodimer (Bishop et al., 1986, Proc. Natl. Acad. Sci. USA 83:4859-4863). The nucleic acid and amino acid sequences of α-galactosidaseA are found in FIGS. 81A (SEQ ID NO:67) and 81B (SEQ ID NO:68). Otheruseful nucleic acid and amino acid sequences of alpha-galactosidase Aare found in U.S. Pat. No.6,329,191.

References teaching how to make alpha-galactosidase A are found in U.S.Pat. Nos. 5,179,023 and 5,658,567 (expression in insect cells), U.S.Pat. No. 5,356,804 (expression and secretion from mammalian cells,including CHO cells), U.S. Pat. No. 5,401,451 (expression in mammaliancells), U.S. Pat. No. 5,580,757 (expression in mammalian cells as afusion protein) and U.S. Pat. No. 5,929,304 (expression in plant cells).Methods for purifying recombinant alpha-galactosidase A are found inU.S. Pat. No. 6,395,884.

References teaching how to use alpha-galactosidase A to treat patientsinclude, but are not limited to, U.S. Pat. No. 6,066,626 (gene therapy)and U.S. Pat. No. 6,461,609 (treatment with the protein). Mutant formsof alpha-galactosidase A that are useful in the methods of the inventioninclude, but are not limited to, those described in U.S. Pat. No.6,210,666.

W. Antibodies

The present invention further comprises a method for the remodeling ofvarious antibody preparations including chimeric antibody preparations,including, chimeric TNFR, chimeric anti-glycoprotein IIb/IIIa, chimericanti-HER2, chimeric anti-RSV, chimeric anti-CD20, and chimeric anti-TNF.Chimeric antibody preparations comprise a human Fc portion from an IgGantibody and the variable regions from a monoclonal antibody specificfor an antigen. Other preparations comprise a receptor, for example the75 kDa TNF receptor, fused to a human IgG Fc portion. These moleculesfurther include Fab fragments comprising light and heavy chains fromhuman and mice. A chimeric TNFR is useful in the treatment ofinflammatory diseases, such as rheumatoid arthritis. Chimericanti-glycoprotein IIb/IIIa is useful in the treatment of cardiacabnormalities, blood clotting, and platelet function disturbances. Achimeric anti-HER2 is useful as a treatment for breast cancer, chimericanti-RSV is useful for the treatment of respiratory syncytial virus,chimeric anti-CD20 is useful for the treatment of Non-Hodgkin'slymphoma, and chimeric anti-TNF is used for treatment of Crohn'sdisease.

While these chimeric antibodies have proved useful in the management ofvaried diseases, administration has to be fairly frequent and at fairlyhigh doses due to the relatively short half-life of a recombinantprotein produced in rodent cells. While a majority of the chimericantibody is human, and therefore regarded as “self” by the immunesystem, they are degraded and destroyed due to non-native glycosylationpatterns. The present invention proposes to repair this problem, greatlyincreasing the efficacy of these novel medicines.

Antibodies and Methods of Their Generation

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as singlechain antibodies and humanized antibodies (Harlow et al., 1999, UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold SpringHarbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; Bird et al., 1988, Science 242:423-426).

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody peptide, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

Monoclonal antibodies directed against full length or peptide fragmentsof a peptide or peptide may be prepared using any well known monoclonalantibody preparation procedures, such as those described, for example,in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold SpringHarbor, N.Y.) and in Tuszynski et al. (1988, Blood, 72:109-115).Quantities of the desired peptide may also be synthesized using chemicalsynthesis technology. Alternatively, DNA encoding the desired peptidemay be cloned and expressed from an appropriate promoter sequence incells suitable for the generation of large quantities of peptide.Monoclonal antibodies directed against the peptide are generated frommice immunized with the peptide using standard procedures as referencedherein.

Nucleic acid encoding the monoclonal antibody obtained using theprocedures described herein may be cloned and sequenced using technologywhich is available in the art, and is described, for example, in Wrightet al. (1992, Critical Rev. in Immunol. 12(3,4):125-168) and thereferences cited therein. Further, the antibody of the invention may be“humanized” using the technology described in Wright et al., (supra) andin the references cited therein, and in Gu et al. (1997, Thrombosis andHematocyst 77(4):755-759).

To generate a phage antibody library, a cDNA library is first obtainedfrom mRNA which is isolated from cells, e.g., the hybridoma, whichexpress the desired peptide to be expressed on the phage surface, e.g.,the desired antibody. cDNA copies of the mRNA are produced using reversetranscriptase. cDNA which specifies immunoglobulin fragments areobtained by PCR and the resulting DNA is cloned into a suitablebacteriophage vector to generate a bacteriophage DNA library comprisingDNA specifying immunoglobulin genes. The procedures for making abacteriophage library comprising heterologous DNA are well known in theart and are described, for example, in Sambrook and Russell (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.).

Bacteriophage which encode the desired antibody, may be engineered suchthat the peptide is displayed on the surface thereof in such a mannerthat it is available for binding to its corresponding binding peptide,e.g., the antigen against which the antibody is directed. Thus, whenbacteriophage which express a specific antibody are incubated in thepresence of a cell which expresses the corresponding antigen, thebacteriophage will bind to the cell. Bacteriophage which do not expressthe antibody will not bind to the cell. Such panning techniques are wellknown in the art and are described for example, in Wright et al.,(supra).

Processes such as those described above, have been developed for theproduction of human antibodies using M13 bacteriophage display (Burtonet al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNA library isgenerated from mRNA obtained from a population of antibody-producingcells. The mRNA encodes rearranged immunoglobulin genes and thus, thecDNA encodes the same. Amplified cDNA is cloned into M13 expressionvectors creating a library of phage which express human antibodyfragments on their surface. Phage which display the antibody of interestare selected by antigen binding and are propagated in bacteria toproduce soluble human immunoglobulin. Thus, in contrast to conventionalmonoclonal antibody synthesis, this procedure immortalizes DNA encodinghuman immunoglobulin rather than cells which express humanimmunoglobulin.

Remodeling Glycans of Antibody Molecules

The specific glycosylation of one class of peptides, namelyimmunoglobulins, has a particularly important effect on the biologicalactivity of these peptides. The invention should not be construed to belimited solely to immunoglobulins of the IgG class, but should also beconstrued to include immunoglobulins of the IgA, IgE and IgM classes ofantibodies.

Further, the invention should not be construed to be limited solely toany type of traditional antibody structure. Rather, the invention shouldbe construed to include all types of antibody molecules, including, forexample, fragments of antibodies, chimeric antibodies, human antibodies,humanized antibodies, etc.

A typical immunoglobulin molecule comprises an effector portion and anantigen binding portion. For a review of immunoglobulins, see Harlow etal., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.,and Harlow et al., 1999, Using Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, NY. The effector portion of theimmunoglobulin molecule resides in the Fc portion of the molecule and isresponsible in part for efficient binding of the immunoglobulin to itscognate cellular receptor. Improper glycosylation of immunoglobulinmolecules particularly in the CH2 domain of the Fc portion of themolecule, affects the biological activity of the immunoglobulin.

More specifically with respect to the immunoglobulin IgG, IgG effectorfunction is governed in large part by whether or not the IgG contains anN-acetylglucosamine (GlcNAc) residue attached at the 4-O position of thebranched mannose of the trimannosyl core of the N-glycan at Asparagine(Asn) 297 in the CH2 domain of the IgG molecule. This residue is knownas a “bisecting GlcNAc.” The purpose of adding bisecting GlcNAc to theN-glycan chains of a natural or recombinant IgG molecule or aIgG-Fc-containing chimeric construct is to optimize Fc immune effectorfunction of the Fc portion of the molecule. Such effector functions mayinclude antibody-dependent cellular cytotoxicity (ADCC) and any otherbiological effects that require efficient binding to FcγR receptors, andbinding to the C1 component of complement. The importance of bisectingGlcNAc for achieving maximum immune effector function of IgG moleculeshas been described (Lifely et al., 1995, Glycobiology 5 (8): 813-822;Jeffris et al., 1990, Biochem. J. 268 (3): 529-537).

The glycans found at the N-glycosylation site at Asn 297 in the CH2domain of IgG molecules have been structurally characterized for IgGmolecules found circulating in human and animal blood plasma, IgGproduced by myeloma cells, hybridoma cells, and a variety of transfectedimmortalized mammalian and insect cell lines. In all cases the N-glycanis either a high mannose chain or a complete (Man3, GlcNAc4, Gal2,NeuAc2, Fuc1) or variably incomplete biantennary chain with or withoutbisecting GlcNAc (Raju et al., 2000, Glycobiology 10 (5): 477-486;Jeffris et al., 1998, Immunological. Rev. 163L59-76; Lerouge et al.,1998, Plant Mol. Biol. 38: 31-48; James et al., 1995, Biotechnology 13:592-596).

The present invention provides an in vitro customized glycosylatedimmunoglobulin molecule. The immunoglobulin molecule may be anyimmunoglobulin molecule, including, but not limited to, a monoclonalantibody, a synthetic antibody, a chimeric antibody, a humanizedantibody, and the like. Specific methods of generating antibodymolecules and their characterization are disclosed elsewhere herein.Preferably, the immunoglobulin is IgG, and more preferably, the IgG is ahumanized or human IgG, most preferably, IgG1.

The present invention specifically contemplates usingβ1,4-mannosyl-glycopeptide β1,4-N-acetylglucosaminyltransferase,GnT-III: EC2.4.1.144 as an in vitro reagent to glycosidically linkN-acetylglucosamine (GlcNAc) onto the 4-O position of the branchedmannose of the trimannosyl core of the N-glycan at Asn 297 in the CH2domain of an IgG molecule. However, as will be appreciated from thedisclosure provided herein, the invention should not be construed tosolely include the use of this enzyme to provide a bisecting GlcNAc toan immunoglobulin molecule. Rather, it has been discovered that it ispossible to modulate the glycosylation pattern of an antibody moleculesuch that the antibody molecule has enhanced biological activity, i.e.,effector function, in addition to potential enhancement of otherproperties, e.g., stability, and the like.

There is provided in the present invention a general method for removingfucose molecules from the Asn(297) N-linked glycan for the purpose ofenhancing binding to Fc-gammaRIIIA, and enhanced antibody-dependentcellular cytotoxicity (see, Shields et al., 2002, J. Biol. Chem.277:26733-26740). The method entails contacting the antibody moleculewith a filcosidase appropriate for the linkage of the fucose molecule(s)on the antibody glycan(s). Alternately, the recombinant antibody can beexpressed in cells that do express fucosyltransferases, such as theLec13 varient of CHO cells. The removal of fucose from the glycan(s) ofthe antibody can be done alone, or in conjunction with other methods toremodel the glycans, such as adding a bisecting GlcNAc. Expression ofantibodies in cells lacking GnT-I may also result in Fc glycans lackingcore fucose, which can be further modified by the present invention.

There is provided in the present invention a general method forintroducing a bisecting GlcNAc for the purpose of enhancing Fc immuneeffector function in any preparation of IgG molecules containingN-linked oligosaccharides in the CH2 domain, typically at Asn 297. Themethod requires that the population of IgG molecules is brought to astate of glycosylation such that the glycan chain is an acceptor forGnT-III. This is accomplished in any one of three ways: 1) by selectionor genetic manipulation of a host expression system that secretes IgGwith N-glycan chains that are substrates for GnT-III; 2) by treatment ofa population of IgG glycoforms with exoglycosidases such that the glycanstructure(s) remaining after exoglycosidase treatment is an acceptor forGnT-III; 3) some combination of host selection and exoglycosidasetreatment as in 1) and 2) above plus successive additions of GlcNAc byGnT-I and GnT-II to create an acceptor for GnT-III.

For example, IgG obtained from chicken plasma contains primarily highmannose chains and would require digestion with one or moreα-mannosidases to create a substrate for addition of GlcNAc to the α1,3mannose branch of the trimannosyl core by GnT-I. This substrate could bethe elemental trimannosyl core, Man3GlcNAc2. Treatment of this corestructure with a combination of GnT-I, GnT-II, and GnT-III usingUDP-GlcNAc as a sugar donor creates Man3GlcNAc5 as shown in FIG. 1. Theorder of action of these glycosyltransferases may be varied to optimizethe production of the desired product. Optionally, this structure canthen be extended by treatment with β1,4 galactosyltransferase. Ifrequired, the galactosylated oligosaccharide can be further extendedusing α2,3- or α2,6-sialyltransferase to achieve a completed biantennarystructure. Using this method biantennary glycan chains can be remodeledas required for the optimal Fc immune effector function of anytherapeutic IgG under development (FIG. 3).

Alternatively, IgG molecules found in the plasma of most animals or IgGwhich is secreted as a recombinant product by most animal cells or bytransgenic animals typically include a spectrum of biantennaryglycoforms including complete (NeuAc2, Gal2, GlcNAc4, Man3, ±Fuc1) (FIG.3) and variably incomplete forms, with or without bisecting GlcNAc (Rajuet al., 2000, Glycobiology 10 (5): 477-486; Jeffris et al., 1998,Immunological Rev. 163: 59-76). To ensure that bisecting GlcNAc ispresent in the entire population of immunoglobulin molecules soproduced, the mixture of molecules can be treated with the followingexoglycosidases, successively or in a mixture: neuraminidase,β-galactosidase, β-hexosaminidase, α-fucosidase. The resultingtrimannosyl core can then be remodeled using glycosyltransferases asnoted above.

In some cases it may be desired to abolish effector function fromexisting antibody molecules. The present invention also includesmodifying the Fc glycans with appropriate glycosidases andglycosyltransferases to eliminate effector function. Also anticipated isthe addition of sugars modified with PEG or other polymers that serve tohinder or abolish binding of Fc receptors or complement to the antibody.

In addition, IgG secreted by transgenic animals or stored as“plantibodies” by transgenic plants have been characterized. An IgGmolecule produced in a transgenic plant having N-glycans that containβ1,2 linked xylose and/or α1,3 linked fucose can be treated withexoglycosidases to remove those residues, in addition to the abovedescribed exoglycosidases in order to create the trimannosyl core or aMan3GlcNAc4 structure, and are then treated with glycosyltransferases toremodel the N-glycan as described above.

The primary novel aspect of the current invention is the application ofappropriate glycosyltransferases, with or without prior exoglycosidasetreatment, applied in the correct sequence to optimize the effectorfunction of the antibody. In one exemplary embodiment, a bisectingGlcNAc is introduced into the glycans of IgG molecules or or otherIgG-Fc-chimeric constructs where bisecting GlcNAc is required. Inanother exemplary embodiment, the core fucose is removed from theglycans of IgG molecules or other IgG-Fc-chimeric constructs.

X. TNF Receptor-IgG Fc Fusion Protein

The nucleotide and amino acid sequences of the 75 kDa human TNF receptorare set forth herein as SEQ ID NO:31 and SEQ ID NO:32, respectively(FIGS. 82A and 82B, respectively). The amino acid sequences of the lightand heavy variable regions of chimeric anti-HER2 are set forth as SEQ IDNO:35 and SEQ ID NO:36, respectively (FIGS. 83A and 83B, respectively).The amino acid sequences of the heavy and light variable regions ofchimeric anti-RSV are set forth as SEQ ID NO:38 and SEQ ID NO:37,respectively (FIGS. 84A and 84B, respectively). The amino acid sequencesof the non-human variable regions of anti-TNF are set forth herein asSEQ ID NO:41 and SEQ ID NO:42, respectively (FIGS. 85A and 85B,respectively). The nucleotide and amino acid sequence of the Fc portionof human IgG is set forth as SEQ ID NO:49 and SEQ ID NO:50 (FIGS. 86Aand 86B, respectively).

A remodeled chimeric ENBREL™ may be administered to a patient selectedfrom the group consisting of a patient having rheumatoid arthritis and apatient having polyarticular-course juvenile arthritis. A remodeledchimeric ENBREL™ may also be administered to an arthritis patient toreduce signs, symptoms, or structural damage in the patient. Preferably,the patient is a human patient.

A remodeled Synagis™ antibody may be administered to a patient toimmunize the patient against infection by respiratory syncytial virus(RSV). A remodeled Synagis™ antibody may also be administered to apatient to prevent or reduce the severity of a lower respiratory tractdisease caused by RSV. Preferably, the patient is a human patient.

Y. MAb Anti-Glycoprotein IIb/IIIa

The amino acid sequences of a murine anti-glycoprotein IIb/IIIa antibodyvariable regions are set forth in SEQ ID NO:52 (murine mature variablelight chain, FIG. 87) and SEQ ID NO: 54 (murine mature variable heavychain, FIG. 88). These murine sequences can be combined with human IgGamino acid sequences SEQ ID NO:51 (human mature variable light chain,FIG. 89), SEQ ID NO: 53 (human mature variable heavy chain, FIG. 90),SEQ ID NO: 55 (human light chain, FIG. 91) and SEQ ID NO: 56 (humanheavy chain, FIG. 92) according to the proceedures found in U.S. Pat.No. 5,777,085 to create a chimeric humanized murine anti-glycoproteinIIb/IIIa antibody. Other anti-glycoprotein IIb/IIIa humanized antibodiesare found in U.S. Pat. No. 5,877,006. A cell line expressing theanti-glycoprotein IIb/IIIa MAb 7E3 can be commercially obtained from theATCC (Manassas, Va.) as accession no. HB-8832.

Indications for Selected Antibodies

A remodeled Reopro™ may be administered to a patient selected from thegroup consisting of a patient undergoing percutaneous coronaryintervention and a patient having unstable angina, wherein the patientis scheduled for percutaneous coronary intervention within 24 hours. Aremodeled Reopro™ may also be administered to a patient undergoingpercutaneous coronary intervention to reduce or prevent a cardiacischemic complication in the patient. Preferably, the patient is a humanpatient.

A remodeled Herceptin™ may be administered to a patient havingmetastatic breast cancer that overexpresses the HER2 protein.Preferably, the patient is a human patient.

A remodeled Remicade™ antibody may be administered to a patient selectedfrom the group consisting of a patient having rheumatoid arthritis, apatient having Crohn's disease, and a patient having fistulizing Crohn'sdisease. A remodeled Remicade™ antibody may also be administered to arheumatoid arthritis patient to reduce signs and symptoms of rheumatoidarthritis in the patient. A remodeled Remicade™ antibody may also beadministered to a Crohn's disease patient to reduce signs and symptomsof Crohn's disease in the patient. Preferably, the patient is a humanpatient.

Z. MAb Anti-CD20

The nucleic acid and amino acid sequences of a chimeric anti-CD20antibody are set forth in SEQ ID NO: 59 (nucleic acid sequence of murinevariable region light chain, FIG. 93A), SEQ ID NO:60 (amino acidsequence of murine variable region light chain, FIG. 93B), SEQ ID NO:61(nucleic acid sequence of murine variable region heavy chain, FIG. 94A)and SEQ ID NO:62 (amino acid sequence of murine variable region heavychain, FIG. 94B). In order to humanize a murine antibody, the TCAE 8(SEQ ID NO:57, FIGS. 95A-95E), which contains the human IgG heavy andlight constant domains, may be conveniently used. By cloning the abovemurine variable region encoding DNA into the TCAE 8 vector according toinstructions given in U.S. Pat. No. 5,736,137, a vector is created (SEQID NO: 58, FIGS. 96A-96E) which when transformed into a mammaliam cellline, expresses a chimeric anti-CD20 antibody. Other humanized anti-CD20antibodies are found in U.S. Pat. No. 6,120,767. A cell line expressingthe anti-CD20 MAb C273 can be commercially obtained from the ATCC(Manassas, Va.) as accession no. HB-9303.

The skilled artisan will readily appreciate that the sequences set forthherein are not exhaustive, but are rather examples of the variableregions, receptors, and other binding moieties of chimeric antibodies.Further, methods to construct chimeric or “humanized” antibodies arewell known in the art, and are described in, for example, U.S. Pat. No.6,329,511 and U.S. Pat. No. 6,210,671. Coupled with the presentdisclosure and methods well known throughout the art, the skilledartisan will recognize that the present invention is not limited to thesequences disclosed herein.

The expression of a chimeric antibody is well known in the art, and isdescribed in detail in, for example, U.S. Pat. No. 6,329,511. Expressionsystems can be prokaryotic, eukaryotic, and the like. Further, theexpression of chimeric antibodies in insect cells using a baculovirusexpression system is described in Putlitz et al. (1990, Bio/Technology8:651-654). Additionally, methods of expressing a nucleic acid encodinga fusion or chimeric protein are well known in the art, and aredescribed in, for example, Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York) andAusubel et al. (1997, Current Protocols in Molecular Biology, Green &Wiley, New York).

Determining the function and biological activity of a chimeric antibodyproduced according to the methods of the present invention is asimilarly basic operation for one of skill in the art. Methods fordetermining the affinity of an antibody by competition assays aredetailed in Berzofsky (J. A. Berzofsky and I. J. Berkower, 1984, inFundamental Immunology (ed. W. E. Paul), Raven Press (New York), 595).Briefly, the affinity of the chimeric antibody is compared to that ofthe monoclonal antibody from which it was derived using aradio-iodinated monoclonal antibody.

A remodeled anti-CD20 antibody may be administered to a patient havingrelapsed or refractory low grade or follicular, CD20-positive, B-cellnon-Hodgkin's lymphoma. Preferably, the patient is a human patient.

VII. Pharmaceutical Compositions

In another aspect, the invention provides a pharmaceutical composition.The pharmaceutical composition includes a pharmaceutically acceptablediluent and a covalent conjugate between a non-naturally-occurring,water-soluble polymer, therapeutic moiety or biomolecule and aglycosylated or non-glycosylated peptide. The polymer, therapeuticmoiety or biomolecule is conjugated to the peptide via an intactglycosyl linking group interposed between and covalently linked to boththe peptide and the polymer, therapeutic moiety or biomolecule.

Pharmaceutical compositions of the invention are suitable for use in avariety of drug delivery systems. Suitable formulations for use in thepresent invention are found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985). For a briefreview of methods for drug delivery, see, Langer, Science 249:1527-1533(1990).

The pharmaceutical compositions may be formulated for any appropriatemanner of administration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268 and 5,075,109.

Commonly, the pharmaceutical compositions are administered parenterally,e.g., intravenously. Thus, the invention provides compositions forparenteral administration which comprise the compound dissolved orsuspended in an acceptable carrier, preferably an aqueous carrier, e.g.,water, buffered water, saline, PBS and the like. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents, detergents and thelike.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably from 5 to 9 and most preferably from 7 and 8.

In some embodiments the peptides of the invention can be incorporatedinto liposomes formed from standard vesicle-forming lipids. A variety ofmethods are available for preparing liposomes, as described in, e.g.,Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980), U.S. Pat. Nos.4,235,871, 4,501,728 and 4,837,028. The targeting of liposomes using avariety of targeting agents (e.g., the sialyl galactosides of theinvention) is well known in the art (see, e.g., U.S. Pat. Nos. 4,957,773and 4,603,044).

Standard methods for coupling targeting agents to liposomes can be used.These methods generally involve incorporation into liposomes of lipidcomponents, such as phosphatidylethanolamine, which can be activated forattachment of targeting agents, or derivatized lipophilic compounds,such as lipid-derivatized peptides of the invention.

Targeting mechanisms generally require that the targeting agents bepositioned on the surface of the liposome in such a manner that thetarget moieties are available for interaction with the target, forexample, a cell surface receptor. The carbohydrates of the invention maybe attached to a lipid molecule before the liposome is formed usingmethods known to those of skill in the art (e.g., alkylation oracylation of a hydroxyl group present on the carbohydrate with a longchain alkyl halide or with a fatty acid, respectively). Alternatively,the liposome may be fashioned in such a way that a connector portion isfirst incorporated into the membrane at the time of forming themembrane. The connector portion must have a lipophilic portion, which isfirmly embedded and anchored in the membrane. It must also have areactive portion, which is chemically available on the aqueous surfaceof the liposome. The reactive portion is selected so that it will bechemically suitable to form a stable chemical bond with the targetingagent or carbohydrate, which is added later. In some cases it ispossible to attach the target agent to the connector molecule directly,but in most instances it is more suitable to use a third molecule to actas a chemical bridge, thus linking the connector molecule which is inthe membrane with the target agent or carbohydrate which is extended,three dimensionally, off of the vesicle surface. The dosage ranges forthe administration of the peptides of the invention are those largeenough to produce the desired effect in which the symptoms of the immuneresponse show some degree of suppression. The dosage should not be solarge as to cause adverse side effects. Generally, the dosage will varywith the age, condition, sex and extent of the disease in the animal andcan be determined by one of skill in the art. The dosage can be adjustedby the individual physician in the event of any counterindications.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achieved bythe use of polymers to conjugate, complex or adsorb the peptide. Thecontrolled delivery may be exercised by selecting appropriatemacromolecules (for example, polyesters, polyaminocarboxymethylcellulose, and protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled release preparations is to incorporate the peptideinto particles of a polymeric material such as polyesters, polyaminoacids, hydrogels, poly (lactic acid) or ethylene vinylacetatecopolymers.

In order to protect peptides from binding with plasma proteins, it ispreferred that the peptides be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methymethacrylate) microcapsules, respectively, or in colloidal drugdelivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such teachings are disclosed in Remington's Pharmaceutical Sciences(16th Ed., A. Oslo, ed., Mack, Easton, Pa., 1980).

The peptides of the invention are well suited for use in targetable drugdelivery systems such as synthetic or natural polymers in the form ofmacromolecular complexes, nanocapsules, microspheres, or beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, liposomes, and resealed erythrocytes. These systems are knowncollectively as colloidal drug delivery systems. Typically, suchcolloidal particles containing the dispersed peptides are about 50 nm-2μm in diameter. The size of the colloidal particles allows them to beadministered intravenously such as by injection, or as an aerosol.Materials used in the preparation of colloidal systems are typicallysterilizable via filter sterilization, nontoxic, and biodegradable, forexample albumin, ethylcellulose, casein, gelatin, lecithin,phospholipids, and soybean oil. Polymeric colloidal systems are preparedby a process similar to the coacervation of microencapsulation.

In an exemplary embodiment, the peptides are components of a liposome,used as a targeted delivery system. When phospholipids are gentlydispersed in aqueous media, they swell, hydrate, and spontaneously formmultilamellar concentric bilayer vesicles with layers of aqueous mediaseparating the lipid bilayer. Such systems are usually referred to asmultilamellar liposomes or multilamellar vesicles (MLVs) and havediameters ranging from about 100 nm to about 4 μm. When MLVs aresonicated, small unilamellar vesicles (SUVS) with diameters in the rangeof from about 20 to about 50 nm are formed, which contain an aqueoussolution in the core of the SUV.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, and phosphatidylethanolamine. Particularly usefulare diacylphosphatidylglycerols, where the lipid moiety contains from14-18 carbon atoms, particularly from 16-18 carbon atoms, and aresaturated. Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.

In preparing liposomes containing the peptides of the invention, suchvariables as the efficiency of peptide encapsulation, lability of thepeptide, homogeneity and size of the resulting population of liposomes,peptide-to-lipid ratio, permeability instability of the preparation, andpharmaceutical acceptability of the formulation should be considered.Szoka, et al, Annual Review of Biophysics and Bioengineering, 9: 467(1980); Deamer, et al., in LIPOSOMES, Marcel Dekker, New York, 1983, 27:Hope, et al., Chem. Phys. Lipids, 40: 89 (1986)).

The targeted delivery system containing the peptides of the inventionmay be administered in a variety of ways to a host, particularly amammalian host, such as intravenously, intramuscularly, subcutaneously,intra-peritoneally, intravascularly, topically, intracavitarily,transdermally, intranasally, and by inhalation. The concentration of thepeptides will vary upon the particular application, the nature of thedisease, the frequency of administration, or the like. The targeteddelivery system-encapsulated peptide may be provided in a formulationcomprising other compounds as appropriate and an aqueous physiologicallyacceptable medium, for example, saline, phosphate buffered saline, orthe like.

The compounds prepared by the methods of the invention may also find useas diagnostic reagents. For example, labeled compounds can be used tolocate areas of inflammation or tumor metastasis in a patient suspectedof having an inflammation. For this use, the compounds can be labeledwith ¹²⁵I, ¹⁴C, or tritium.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

The materials and methods used in the experiments presented in thisExample are now described.

A. General Procedures

1. Preparation of CMP-SA-PEG

This example sets forth the preparation of CMP-SA-PEG.

Preparation of2-(benzyloxycarboxamido)-glycylamido-2-deoxy-D-mannopyranose.N-benzyloxycarbonyl-glycyl-N-hydroxysuccinimide ester (3.125 g, 10.2mmol) was added to a solution containing D-mannosamine-HCl (2 g, 9.3mmol) and triethylamine (1.42 mL, 10.2 mmol) dissolved in MeOH (10 mL)and H₂O (6 mL). The reaction was stirred at room temperature for 16hours and concentrated using rotoevaporation. Chromatography (silica,10%MeOH/CH₂Cl₂) yielded 1.71 g (50% yield) of product as a white solid:R_(f)=0.62 (silica; CHCl₃:MeOH:H₂O, 6/4/1); ¹H NMR (CD₃OD, 500 MHz) δ3.24-3.27 (m, 2H), 3.44 (t, 1H), 3.55 (t, 1H), 3.63-3.66 (m, 1H),3.76-3.90 (m, 6H), 3.91 (s, 2H), 4.0 (dd, 2 H), 4.28 (d, 1H, J=4.4),4.41 (d, 1H, J=3.2), 5.03 (s, 1H), 5.10 (m, 3H), 7.29-7.38(m, 10H).

Preparation of5-(N-benzyloxycarboxamido)glycylamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosuronate.2-(N-Benzyloxycarboxamido) glycylamide-2-deoxy-D-mannopyranose (1.59 g,4.3 mmol) was dissolved in a solution of 0.1 M HEPES (12 mL, pH 7.5) andsodium pyruvate (4.73 g, 43 mmol). Neuraminic acid aldolase (540 U ofenzyme in 45 mL of a 10 mM phosphate buffered solution containing 0.1 MNaCl at pH 6.9) and the reaction mixture was heated to 37° C. for 24 hr.The reaction mixture was then centrifuged and the supernatant waschromatographed (C18 silica, gradient from H₂O (100%) to 30%MeOH/water). Appropriate fractions were pooled, concentrated and theresidue chromatographed (silica, gradient from 10% MeOH/CH₂Cl₂ toCH₂Cl₂/MeOH/H₂O6/4/1). Appropriate fractions were collected,concentrated and the residue resuspended in water. After freeze-drying,the product (1.67 g, 87% yield) was obtained as a white solid:R_(f)=0.26 (silica, CHCl₃/MeOH/H₂O6/4/1); ¹H NMR (D₂O, 500 MHz) δ 1.82(t, 1H), 2.20 (m, 1H), 3.49 (d, 1H), 3.59(dd, 1H), 3.67-3.86 (m, 2H),3.87(s, 2H), 8.89-4.05 (m, 3H), 5.16 (s, 2H), 7.45 (m, 5H).

Preparation of5-glycylamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosuronate.5-(N-Benzyloxycarboxamido)glycylamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosuronate(1.66 g , 3.6 mmol) was dissolved in 20 mL of 50% water/methanol. Theflask was repeatedly evacuated and placed under argon and then 10% Pd/C(0.225 g) was added. After repeated evacuation, hydrogen (about 1 atm)was then added to the flask and the reaction mixture stirred for 18 hr.The reaction mixture was filtered through celite, concentrated by rotaryevaporation and freeze-dried to yield 1.24 g (100% yield) of product asa white solid: R_(f)=0.25 (silica, IPA/H₂O/NH₄OH 7/2/1); ¹H NMR (D₂O,500 MHz) δ 1.83 (t, 1H, J=9.9), 2.23 (dd, 1H, J=12.9, 4.69), 3.51-3.70(m, 2H), 3.61(s, 2H), 3.75-3.84 (m, 2H), 3.95-4.06(m, 3H).

Preparation ofcytidine-5′-monophosphoryl-[5-(N-fluorenylmethoxy-carboxamido)glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate].A solution containing5-glycylamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosuronate(0.55 g, 1.70 mmol) dissolved in 20 mL H₂O was added to a solution ofTris (1.38 g, 11.4 mmol), 1 M MgCl₂ (1.1 mL) and BSA (55 mg). The pH ofthe solution was adjusted to 8.8 with 1M NaOH (2 mL) and CTP-2Na⁺(2.23g, 4.2 mmol) was added. The reaction mixture pH was controlled with a pHcontroller which delivered 1 M NaOH as needed to maintain pH 8.8. Thefusion protein (sialyltransferase/CMP-neuraminic acid synthetase) wasadded to the solution and the reaction mixture was stirred at roomtemperature. After 2 days, an additional amount of fusion protein wasadded and the reaction stirred an additional 40 hours. The reactionmixture was precipitated in EtOH and the precipitate was washed 5 timeswith cold EtOH to yield 2.3 grams of a white solid. About 1.0 g of thecrude product was dissolved in 1,4 dioxane (4 mL), H₂O (4 mL) andsaturated NaHCO₃ (3 mL) and a solution of FMOC-Cl (308 mg, 1.2 mmol)dissolved in 2 ml dioxane was added dropwise. After stirring for 16 hrat room temperature, the reaction mixture was concentrated to about 6 mLby rotary evaporation and purified using chromatography (C18 silica,gradient 100% H₂O to 30% MeOH/H₂O). Appropriate fractions were combinedand concentrated. The residue was dissolved in water and freeze-dried toyield 253 mg of a white solid: R_(f)=0.50 (silica, IPA/H₂O/NH₄OH 7/2/1);¹H NMR (D₂O, 500 MHz) δ 1.64 (dt, 1H, J=12.0, 6.0), 2.50 (dd, 1H,J=13.2, 4.9), 3.38 (d, J=9.67, 1H), 3.60 (dd, J=11.65, 6.64, 1H), 3.79(d, J=4.11, 1H), 3.87 (dd, J=12.24, 1.0, 1H), 3.97 (m, 2H), 4.07 (td,J=10.75, 4.84, 1H), 4.17 (dd, J=10.68, 1.0, 1 H), 4.25 (s, 2H), 4.32 (t,J=4.4, 1H), 4.37 (t, J=5.8 1H), 4.6-4.7 (m, obscured by solvent peak),5.95 (d, J=4, 1 H), 6.03 (d, J=7.4, 1H), 7.43-7.53 (m, 3H), 7.74 (m,2H), 7.94 (q, J=7, 3H). MS (ES); calc. for C₃₅H₄₂N₅O₁₈P ([M-H]⁻), 851.7;found 850.0.

Preparation ofcytidine-5′-monophosphoryl-(5-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate).Diisopropylamine (83 uL, 0.587 μmol) was added to a solution ofcytidine-5′-monophosphoryl-[5-(N-fluorenyl-methoxycarboxamido)glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate](100 mg, 0.117 mmol) dissolved in water (3 mL) and methanol (1 mL). Thereaction mixture was stirred 16 hr at room temperature and the reactionmethanol removed from the reaction mixture by rotary evaporation. Thecrude reaction mixture was filtered through a C18 silica gel columnusing water and the efluant was collected and freeze-dried to yield (87mg, 100%) of product as a white solid: R_(f)=0.21 (silica, IPA/H₂O/NH₄OH7/2/1); ¹H NMR (D₂O, 500 MHz) δ 1.66 (td, 1H, J=5.3), 2.50 (dd, 1H,J=13.2, 4.6), 3.43 (d, J=9.58, 1H), 3.63 (dd, J=11.9, 6.44, 1H), 3.88(dd, J=11.8, 1.0, 1H), 3.95 (td, J=9.0, 2.3, 1H), 4.10 (t, J=10.42, 1H),4.12 (td, J=10.34, 4.66, 1 H), 4.18 (d, J=10.36, 1H), 4.24 (m, 2H), 4.31(t, J=4.64, 1H), 4.35 (t, 1H), 6.00 (d, J=4.37, 1 H), 6.13 (d, J=7.71,1H), 7.98 (d, J=7.64, 1H). MS (ES); calc. for C₂₁H₃₂N₅O₁₁P ([M-H]⁻),629.47; found 627.9.

Preparation ofcytidine-5′-monophosphoryl-[5-(N-methoxy-polyoxyethylene-(1kDa)-3-oxypropionamido)-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate].Benzyltriazol-1-yloxy-tris(dimethylamino)-phosphoniumhexafluorophosphate (BOP, 21 mg, 48 μmol) was added to a solution ofmethoxypolyoxyethylene-(1 kDa average molecular weight)-3-oxypropionicacid (48 mg, 48 μmol) dissolved in anhydrous DMF (700 μL) andtriethylamine (13 μL, 95 μmol). After 30 min, a solution containingcytidine-5′-monophosphoryl-(5-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate)(30 mg, 48 μmol), water (400 μL) and triethylamine (13 μL, 95 μmol) wasadded. This solution was stirred 20 min at room temperature and thenchromatographed (C18 silica, gradient of methanol/water). Appropriatefractions were collected, concentrated, the residue dissolved in waterand freeze-dried to afford 40 mg (50% yield) of a white solid:R_(f)=0.36 (silica, IPA/H₂O/NH₄OH 7/2/1); ¹H NMR (D₂O, 500 MHz) δ 1.66(td, 1H, J=5.3), 2.50 (dd, 1H, J=13.2, 4.6), 2.64 (t, J=5.99, 3H) 3.43(d, J=9.58, 1H), 3.63 (m, 1H), 3.71 (s, 70H), 3.79 (m, obscured by 3.71peak), 3.82 (t, J=6.19, 1H) 3.88 (dd, J=11.8, 1.0, 1H), 3.95 (td, J=9.0,2.3, 1H), 3.98 (t, J=5.06, 1H), 4.12 (td, J=10.34, 4.66, 1 H), 4.18 (d,J=10.36, 1H), 4.23 (d, J=4.85, 2H), 4.31 (t, J=4.64, 1H), 4.35 (t, 1H),6.00 (d, J=4.55, 1 H), 6.13 (d, J=7.56, 1H), 7.98 (d, J=7.54, 1H). MS(MALDI), observe [M-H]; 1594.5, 1638.5, 1682.4, 1726.4, 1770.3, 1814.4,1858.2, 1881.5, 1903.5, 1947.3.

Preparation ofcytidine-5′-monophosphoryl-[5-(N-methoxy-polyoxyethylene-(10kDa)-oxycarboxamido)-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate].Cytidine-5′-monophosphoryl-(5-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate)(2.5 mg, 4 μmol) and water (180 μL) was added to a solution of(Methoxypolyoxyethylene-(10 kDa, average molecularweight)-oxycarbonyl-(N-oxybenzotriazole) ester (40 mg, 4 μmol) inanhydrous DMF (800 μL) containing triethylamine (1.1 μL, 8 μmol) and thereaction mixture stirred for 1 hr at room temperature. The reactionmixture was then diluted with water (8 mL) and was purified by reversedphase flash chromatography (C18 silica, gradient of methanol/water).Appropriate fractions were combined, concentrated, the residue dissolvedin water and freeze-dried yielding 20 mg (46% yield) of product as awhite solid: R_(f)=0.35 (silica, IPA/H₂O/NH₄OH 7/2/1); ¹H NMR (D₂O, 500MHz) δ 1.66 (td, 1H), 2.50 (dd, 1H), 2.64 (t, 3H) 3.55-3.7 (m, obscuredby 3.71 peak), 3.71 (s, 488H), 3.72-4.0 (m, obscured by 3.71 peak), 4.23(m, 3H), 4.31 (t, 1H), 4.35 (t, 1H), 6.00 (d, J=4.77, 1 H), 6.12 (d,J=7.52, 1H), 7.98 (d, J=7.89, 1H). MS (MALDI), observe [M−CMP+Na];10780.

2. Preparation of CMP-SA-PEG II

This example sets forth the general procedure for making CMP-SA-PEG, andspecific procedures for making CMP-SA-PEG (1 kDa) and CMP-SA-PEG (20kDa).

General procedures PreparingCytidine-5′-monophosphoryl-(5-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate).Cytidine-5′-monophosphoryl-(5-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate(870 mg, 1.02 mmol) was dissolved in 25 mL of water and 5.5 mL of 40 wt% dimethylamine solution in H₂O was added. The reaction was stirred for1 hr and the excess dimethyl amine was then removed by rotaryevaporation. The aqueous solution was filtered through a C-18 silica gelcolumn and the column was washed with water. The eluants were combinedand lyophilized to afford 638 mg (93%) of a white solid. R_(f)=0.10(silica, IPA/H₂O/NH₄OH; 7/2/1). ¹H NMR (D₂O, 500 MHz) δ 1.66 (td, 1H,J=5.3), 2.50 (dd, 1H, J=13.2, 4.6), 3.43 (d, J=9.48, 1H), 3.63 (dd,J=11.9, 6.44, 1H), 3.88 (dd, J=11.8, 1.0, 1H), 3.95 (td, J=9.0, 2.3,1H), 4.10 (t, J=10.42, 1H), 4.12 (td, J=10.34, 4.66, 1 H), 4.18 (d,J=10.36, 1H), 4.24 (m, 2H), 4.31 (t, J=4.64, 1H), 4.35 (t, 1H), 6.00 (d,J=4.37, 1 H), 6.13 (d, J=7.71, 1H), 7.98 (d, J=7.64, 1H). MS (ES); calc.for C₂₁H₃₂N₅O₁₁P ([M-H]⁻), 629.47; found 627.9.

General procedures for Preparing CMP-SA-PEG usingmPEG-(p-nitrophenol)carbonate.Cytidine-5′-monophosphoryl-(5-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate)(175 mg, 0.259 mMol) was dissolved in a mixture of water, pH 8.5, andDMF or THF (in a ratio of 1:2). The mPEG-nitrophenol carbonate (2 to 20kDa mPEG's) (0.519 mMole) was added in several portions over 8 hr atroom temperature and the reaction mixture was stirred at roomtemperature for 3 days. When complete, water (40 ml) and 1.5 ml of NH₄OH(29% aqueous solution) were added. The yellow reaction mixture wasstirred for another 2 hr and then concentrated by rotary evaporation.The reaction mixture was then diluted with water (pH 8.5) to about 500ml volume and was purified by reversed phase flash chromatography(Biotage 40M, C18 silica column) with a gradient of methanol/water.Appropriate fractions were combined and concentrated to afford theproducts as white solids. R_(f) (silica; 1-propanol/water/29% NH₄OH;7/2/1); (2 kDa PEG)=0.31; (5 kDa PEG)=0.33; (10 kDa PEG)=0.36; (20 kDaPEG)=0.38 (TLC silica, IPA/H₂O/NH₄OH 7/2/1); MS (MALDI), observe[M−CMP+Na]; (2 kDa)=2460; (5 kDa)=5250; (10 kDa)=10700; (20 kDa)=22500.

Preparation ofCytidine-5′-monophosphoryl-[5-(N-fluorenylmethoxycarboxamido)-glycylamido3,5-dideoxy-β-D-glycero-D-galacto2-nonulopyranosuronate]. Solium pyruvate (2.4g, 218 mmol), HEPES buffer (0.25 M, pH 7.34) and 1.0 g (22 mmol) ofFmoc-glycylmannosamide were mixed in a 150 mL polycarbonate bottle. Aneuraminic acid aldolase solution (19 mL, ˜600 U) was then added and thereaction mixture was incubated at 30° C. on an orbital shaker. After 23hours, Thin layer chromatography (TLC) indicated that approximately 75%conversion to product had occurred. The CTP (1.72 g, 33 mmol) and 0.1 Mof MnCl₂ (6 mL) were then added to the reaction mixture. The pH wasadjusted to 7.5 with 1 M NaOH (5.5 mL) and a solution containingCMP-neuraminic acid synthetase (Neisseria) was added (25 mL, 386 U). Thereaction was complete after 24 hrs and the reaction mixture waschromatographed (C-18 silica, gradient from H₂O (100%) to 10% MeOH/H₂O).Appropriate fractions were recombined, concentrated and lyophilized toafford a white solid, R_(f) (IPA/ H₂O/NH₄OH, 7/2/1)=0.52. ¹H NMR (D₂O,500 MHz) δ 1.64 (dt, 1H, J=12.0, 6.0), 2.50 (dd, 1H, J=13.2, 4.9), 3.38(d, J=9.67, 1H), 3.60 (dd, J=11.65, 6.64, 1H), 3.79 (d, J=4.11, 1H),3.87 (dd, J=12.24, 1.0, 1H), 3.97 (m, 2H), 4.07 (td, J=10.75, 4.84, 1H),4.17 (dd, J=10.68, 1.0, 1 H), 4.25 (s, 2H), 4.32 (t, J=4.4, 1H), 4.37(t, J=5.8 1H), 4.6-4.7 (m, obscured by solvent peak), 5.95 (d, J=4, 1H), 6.03 (d, J=7.4, 1H), 7.43-7.53 (m, 3H), 7.74 (m, 2H), 7.94 (q, J=7,3H) MS (ES); calc. for C₃₅H₄₂N₅O₁₈P ([M-H]⁻), 850.7; found 850.8.

Preparation of Cytidine-5′-monophosphoryl-[5-(N-methoxypolyoxyethylene(1kDa)-3-oxypropionamido)-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate].Methoxypolyoxyethylene-(1 kDa average molecularweight)-3-oxypropionate-N-succinimidyl ester (52 mg, 52 μmol) dissolvedin anhydrous DMF (450 μL) and triethylamine (33 μL, 238 μmol).Cytidine-5′-monophosphoryl-(5-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate)(30 mg, 48 μmol) was added as a solid. Water, pH 8 (330 μL) was addedand after 30 min, an additional 28 mg of NHS-activated PEG was added.After an additional 5 min, the reaction mixture was chromatographed(C-18 silica, gradient of methanol/water), and appropriate fractionswere concentrated to afford 32 mg (40% yield) of a white solid,R_(f)=0.31 (silica, IPA/H₂O/NH₄OH 7/2/1); ¹H NMR (D₂O, 500 MHz) δ 1.66(td, 1H, J=5.3), 2.50 (dd, 1H, J=13.2, 4.6), 2.64 (t, J=5.99, 3H) 3.43(d, J=9.58, 1H), 3.63 (m, 1H), 3.71 (s, 70H), 3.79 (m, obscured by 3.71peak), 3.82 (t, J=6.19, 1H) 3.88 (dd, J=11.8, 1.0, 1H), 3.95 (td, J=9.0,2.3, 1H), 3.98 (t, J=5.06, 1H), 4.12 (td, J=10.34, 4.66, 1 H), 4.18 (d,J=10.36, 1H), 4.23 (d, J=4.85, 2H), 4.31 (t, J=4.64, 1H), 4.35 (t, 1H),6.00 (d, J=4.55, 1 H), 6.13 (d, J=7.56, 1H), 7.98 (d, J=7.54, 1H). MS(MALDI), observe [(M−CMP)-H]; 1506.4, 1550.4, 1594.5, 1638.5, 1682.4,1726.4, 1770.3, 1814.4, 1858.2.

Preparation ofCytidine5′-monophosphoryl-{5-[N-(2,6-dimethoxypolyoxyethylene-(20kDa)-3oxypropionamidyl-lysylamido]-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate}.The 2,6-Di-[methoxypolyoxyethylene-(20 kDa average molecularweight)-3-oxypropionamidyl]-lysylamido-N-succinimidyl ester (367 mg, 9μmol) was dissolved in anhydrous THF (7 mL) and triethylamine (5 μL, 36μmol).Cytidine-5′-monophosphoryl-(5-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate)(30 mg, 48 μmol) was dissolved in 1.0 mL of water, and added to thereaction mixture. The reaction was stirred for 4 hours at roomtemperature and was then chromotographed (HPLC, Waters Xterra RP8,gradient from water/NH4OH, 100% to 20% methanol/water/NH₄OH at 1 mL/min)to afford a white solid with a R_(t)=22.8 min. MS (MALDI), observe[(M-CMP)-H]; 43027.01 (40,000-45,500).

3. Preparation of UDP-Gal-PEG.

This example sets forth the general procedure for making UDP-Gal-PEG.

Methoxypolyoxyethylenepropionate N-hydroxysuccinimide ester (mPEG-SPA,MW 1,000) 348 mg in THF (0.5 mL) was added to a solution of 25 mg ofgalactosamine-1-phosphate in 1 ml of water, followed by the addition of67 μL triethylamine. The resulting mixture was stirred at roomtemperature for 17 hr. Concentration at reduce pressures provided acrude reaction mixture which was purified by chromatography (C-18silica, using a step gradient of 10%, 20%, 30%, 40% aqueous MeOH) toafford 90 mg (74%) of product after the appropriate fractions werecombined and concentrated to dryness. R_(f)=0.5 (silica,Propanol/H₂O/NH₄OH 30/20/2); MS(MALDI), observed 1356, 1400, 1444, 1488,1532, 1576, 1620.

[α-1-(Uridine-5′-diphosphoryl)]-2-deoxy-2-(methoxypolyoxyethylene-propionoylamido-1kDa)-α-D-galactosamine. The2-deoxy-2-(methoxy-polyoxyethylenepropionoylamido-1kDa)-α-1-monophosphate-D-galactosamine (58 mg) was dissolved in 6 mL ofDMF and 1.2 mL of pyridine. UMP-morpholidate (60 mg) was then added andthe resulting mixture was stirred at 70° C. for 48 hr. Afterconcentration, the residue was chromatographed (C1 8-silica, using astep gradient of 10%, 20%, 30%, 40%, 50%, 80% MeOH) to yield 50 mg ofproduct after concentration of the appropriate fractions. R_(f)=0.54(silica, propanol/H₂O/NH₄OH 30/20/2). MS(MALDI); Observed 1485, 1529,1618, 1706.

[α-1-(Uridine-5′-diphosphoryl)]-6-deoxy-6-(methoxypolyoxyethylene-amino-2kDa)-α-D-galactose.[α-1-(Uridine-5′-diphosphoryl)]-6-carboxaldehyde-α-D-galactose (10 mg)was dissolved in 2 mL of 25 mM sodium phosphate buffer (pH 6.0) andtreated with methoxypolyethyleneglycol amine (MW 2,000, 70 mg) and then25 μL of 1M NaBH₃CN solution at 0° C. The resulting mixture was frozenat −20° C. for three days. The reaction mixture was chromatographed(HPLC, Water Xterra P8) using 0.015 M NH₄OH as mobile phase A and MeOHas mobile phase B as eluent at the speed of 1.0 mL/min. The product wascollected, an concentrated to yield a solid; R_(t)=9.4 minutes.R_(f)=0.27(silica, EtOH/H₂O 7/3).

[α-1-(Uridine-5′-diphosphoryl)]-6-amino-6-deoxy-α-D-galactose. Ammoniumacetate 15 mg was added to a solution of[α-1-(Uridine-5′-diphosphoryl)]-6-carboxaldehyde-α-D-galactopyranoside(10 mg) in sodium phosphate buffer (pH 6.0). A solution of (25 μL) 1MNaBH₃CN was then added and the mixture was stirred for 24 hr. Thesolution was concentrated and the residue was chromotographed (sephadexG₁₀) to afford 10 mg of a white solid, R_(f)=0.62 (silica, EtOH/0.1 MNH₄Ac).

[α-1-(Uridine-5-diphosphoryl)]-6-deoxy-6-(methoxypolyoxyethylenepropionoylamido, ˜2 kDa)-α-D-galactopyranoside.[α-1-(Uridine-5′-diphosphoryl)]-6-amino-6-deoxy-α-D-galactopyranoside (5mg) was dissolved in 1 mL of H₂O. Thenmethoxypolyetheneglycolpropionoyl-NHS ester (MW ˜2,000, 66 mg) wasadded, followed by 4.6 μL triethylamine. The resulting mixture wasstirred at room temperature overnight, and then purified on HPLC (C-8silica) to afford the product, R_(t)=9.0 min.

[α-1-(Uridine-5′-diphosphoryl)]-6-deoxy-6-(methoxypolyoxyethylenecarboxamido,2 kDa)-α-D-galactopyranoside.[α-1-(Uridine-5′-diphosphoryl)]-6-amino-6-deoxy-α-D-galactopyranoside(10 mg) was mixed with methoxypolyethyleneglycolcarboxy-HOBT (MW 2000,67 mg) in 1 mL of H₂O, followed by the addition ofEDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride 6.4 mgand 4.6 μL triethylamine. The resulting mixture was stirred at roomtemperature 24 hr. The mixture was chromatographed (C-8 silica) toafford the product.

4. Preparation of UDP-GlcNAc-PEG

This example sets forth the general procedure for making UDP-GlcNAc-PEG.On the left side of scheme 17, the protected amino sugardiphospho-nucleotide is oxidized to form an aldehyde at the 6-positionof the sugar. The aldehyde is converted to the corresponding primaryamine by formation and reduction of the Schiff base. The resultingadduct is contacted with the p-nitrophenol carbonate of m-PEG, whichreacts with the amine, binding the m-PEG to the saccharide nucleus viaan amide bond. On the right side of scheme 17 at the top, the protectedamino sugar diphospho-nucleotide is treated with a chemical oxidant toform a carboxyl group at the 6-carbon of the sugar nucleus. The carboxylgroup is activated and reacted with m-PEG amine, binding the m-PEG tothe saccharide nucleus via an amide bond. On the right side of scheme 17at the bottom the reactions are substantially similar to that on the topright, with the exception that the starting sugar nucleotide iscontacted with an oxidizing enzyme, such as a dehydrogenase, rather thana chemical oxidant.

5. Preparation of UDP-GalNAc-PEG

This example (scheme 18) sets forth the general procedure for makingUDP-GalNAc-PEG. The reaction set forth above originates with a sugardiphospho-nucleotide, in which R is either a hydroxyl 1 or a protectedamine 2. In step a, the starting sugar is treated with a mixture of anoxidase and a catalase, converting the 6-postion of the sugar into analdehyde moiety (3 and 4). In step c, the aldehyde is converted to thecorresponding amine (7 and 8) by formation and reduction of a Schiffbase. In step e, the amine is optionally treated with an activated m-PEGderivative, thereby acylating the amine to produce the correspondingm-PEG amide (11 and 13). Alternatively, in step f, the amine iscontacted with an activated m-PEG species, such as a m-PEG active ester,thereby forming the corresponding m-PEG amide (12 and 14). In step b,the starting material is also treated with a catalase and oxidase,completely oxidizing the hydroxymethyl moiety, forming a carboxyl groupat the 6-position. In step d, the carboxyl moiety is activated andsubsequently converted to a m-PEG adduct (9 and 10) by reaction with am-PEG amine intermediate. This is shown in scheme 18.

The amino-sugar phosphate is contacted with a m-PEG N-hydroxysuccinimide active ester, thereby forming the correspondingsugar-PEG-amide. The amide is contacted with UMP-morpholidate to formthe corresponding active sugar diphospho-nucleotide.

6. Synthesis of CMP-SA-Levulinate

This example sets forth the procedure for the synthesis ofCMP-SA-levulinate.

Preparation of 2-levulinamido-2-deoxy-D-mannopyranose.Isobutyichloroformate (100 μL, 0.77 mmol) was added dropwise to asolution of levulinic acid (86 μL, 0.84 mmol), anhydrous THF (3 mL) andtriethylamine (127 μL, 0.91 mmol). This solution was stirred for 3 hoursat room temperature and was then added dropwise to a solution containingD-mannosamine hydrochloride (151 mg, 0.7 mmol), triethylamine (127 μL,0.91 mmol), THF (2 mL) and water (2 mL). The reaction mixture wasstirred 15 hours and then concentrated to dryness by rotary evaporation.Chromatography (silica, step gradient of 5-15% MeOH/CH₂Cl₂) was used toisolate the product yielding 0.156 g (73% yield) of a white solid:R_(f)=0.41 (silica, CHCl₃/MeOH/water 6/4/1); ¹H NMR (D₂O, 500 MHz) δ2.23 (s, 3H), 2.24 (s, 3H), 2.57(td, J=6.54, 3.68, 2H) 2.63 (t, J=6.71,2H), 2.86-2.90 (m, 4H), 3.42 (m, 1H), 3.53 (t, J=9.76, 1H), 3.64 (t,J=9.43, 1H), 3.80-3.91 (m, 4H), 4.04 (dd, J=9.79, 4.71, 1H), 4.31 (dd,J=4.63, 1.14, 1H), 4.45 (dd, J=4.16, 1.13, 1H), 5.02 (d, J=1.29, 1H),5.11(s, J=1.30, 1H), MS (ES); calculated for C₁₁H₁₉NO₇, 277.27; found[M+1] 277.9.

Preparation of5-levulinamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosuronate.Sodium pyruvate (0.616 g, 5.6 mmol) and N-acetylneuraminic acid aldolase(50 U) was added to a solution of 2-levulinamido-2-deoxy-D-mannopyranose(0.156 g, 0.56 mmol) in 0.1 M HEPES (pH 7.5). The reaction mixture washeated to 37° C. for 20 hours and after freezing. The reaction mixturewas then filtered through C1 8 silica, frozen and freeze-dried. Thecrude solid was purified using flash chromatography (silica, first using10-40% MeOH/CH₂Cl₂ and then CH₂Cl₂/MeOH/H₂O 6/4/0.5). Appropriatefractions were combined and concentrated yielding 45 mg (80% yield) of awhite solid: R_(f)=0.15 (silica, CHCl₃/MeOH/water 6/4/1); ¹H NMR (D₂O,500 MHz) δ 1.82 (t, J=11.9, 1H), 2.21 (dd, J=13.76, 4.84, 1H), 2.23 (s,3H), 2.57 (app q, J=6.6, 2H), 2.86-2.95 (m, 2H), 3.15-3.18 (m, 1H),3.28-3.61 (complex, 1H), 3.60 (dd, J=11.91, 6.66, 1H), 3.75 (td, J=6.65,2.62, 1H), 3.84 (dd, J=11.89, 2.65, 1 H), 3.88-4.01 (complex, 2H), 4.04(td, J=11.18, 4.67, 1H), MS (ES); calculated for Cl₁₄H₂₃NO₁₀, 365.33;found ([M-1]⁻), 363.97.

Preparation ofcytidine-5′-monophosphoryl-(5-levulinamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate).5-Levulinamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosuronate(50 mg, 137 μmol) was dissolved in 2 mL of 100 mM HEPES pH 7.5 bufferand 1 M MnCl₂ (300 μL, 300 μmol) was added. CTP-2Na⁺(79 mg, 1.5 μmol)was dissolved in 5 mL HEPES buffer and was added to the sugar. Thesialyltransferase/CMP-neuraminic acid synthetase fusion enzyme (11 U)was added and the reaction mixture stirred at room temperature for 45hours. The reaction mixture was filtered through a 10,000 MWCO filterand the filtrate, which contained the product of the reaction, was useddirectly without further purification: R_(f)=0.35 (silica,IPA/water/NH₄OH 7/2/1).

B. Glycoconjugation and GlycoPEGylation of Peptides

α-Protease Inhibitor (α-Antitrypsin)

7. Sialylation of Recombinant GlycoproteinsAntithrombin III, Fetuin andα1-Antitrypsin

This example sets forth the preparation of sialylated forms of severalrecombinant peptides.

Sialylation of Recombinant Glycoproteins Using ST3Gal III. Severalglycoproteins were examined for their ability to be sialylated byrecombinant rat ST3Gal III. For each of these glycoproteins, sialylationwill be a valuable process step in the development of the respectiveglycoproteins as commercial products.

Reaction Conditions. Reaction conditions were as summarized in Table 11.The sialyltransferase reactions were carried out for 24 hour at atemperature between room temperature and 37°. The extent of sialylationwas established by determining the amount of ¹⁴C-NeuAc incorporated intoglycoprotein-linked oligosaccharides. See Table 11 for the reactionconditions for each protein.

TABLE 11 Reaction conditions. Protein Protein ST CMP- Total Conc. (mU/ST/Protein NeuAc of Protein Source (mg) (mg/ml) mL) (mU/mg) “cycle”¹ATIII Genzyme 8.6 4.3 210 48 cycle Transgenics ATIII Genzyme 860 403 5312 cycle Transgenics Asialo- Sigma 0.4 105 20 13 10 mM fetuin asilao-PPL 0.4 0.5 20 20 20 mM AAAT ¹“Cycle” refers to generation of CMP-NeuAc“in situ” enzymatically using standard conditions as described inspecification (20 mM NeuAc and 2 mM CMP). The buffer was 0.1 M HEPES, pH7.5.

The results presented in Table 12 demonstrate that a remarkable extentof sialylation was achieved in every case, despite low levels of enzymeused. Essentially, complete sialylation was obtained, based on theestimate of available terminal galactose. Table 12 shows the relults ofthe sialylation reactions. The amount of enzyme used per mg of protein(mU/mg) as a basis of comparison for the various studies. In several ofthe examples shown, only 7-13 mU ST3Gal III per mg of protein wasrequired to give essentially complete sialylation after 24 hours.

TABLE 12 Analytical results Terminal NeuAc Gal¹ Incorp.² % Other ProteinSource mol/mol mol/mol Rxn³ characterization ATIII⁴ Genzyme 102 104 117None Transgenics ATIII⁴ Genzyme 102 1.3 108 SDS-gels: proteinTransgenics purity FACs: carbohydrate glycoforms Asialo- Sigma 802 905116 None fetuin asilao- PPL 7 7.0 100 SDS-gels: protein AAAT⁵ purity¹Terminal (exposed) Gal content on N-linked oligosaccharides determinedby supplier, or from literatures values (fetuin, asialo-AAAT). ²NeuAcincorporated determined by incorporation of 14C-NeuAc after separationfrom free radiolabeled precursors by gel filtration. ³The % Rxn refersto % completion of the reaction based on the terminal Gal content as atheoretical maximum. ⁴Antithrombin III. ⁵α1 Antitrypsin.

These results are in marked contrast to those reported in detailedstudies with bovine ST6Gal I where less than 50 mU/mg protein gave lessthan 50% sialylation, and 1070 mU/mg protein gave approximately 85-90%sialylation in 24 hours. Paulson et al. (1977) J. Biol. Chem. 252:2363-2371; Paulson et al. (1978) J. Biol. Chem. 253: 5617-5624. A studyof rat α2,3 and α2,6 sialyltransferases by another group revealed thatcomplete sialylation of asialo-AGP required enzyme concentrations of150-250 mU/mg protein (Weinstein et al. (1982) J. Biol. Chem. 257:13845-13853). These earlier studies taken together suggested that theST6Gal I sialyltransferase requires greater than 50 mU/mg and up to 150mU/mg to achieve complete sialylation.

This Example demonstrates that sialylation of recombinant glycoproteinsusing the ST3 Gal III sialyltransferase required much less enzyme thanexpected. For a one kilogram scale reaction, approximately 7,000 unitsof the ST3Gal III sialyltransferase would be needed, instead of100,000-150,000 units that earlier studies indicated. Purification ofthese enzymes from natural sources is prohibitive, with yields of only1-10 units for a large scale preparation after 1-2 months work. Assumingthat both the ST6Gal I and ST3 Gal III sialyltransferases are producedas recombinant sialyltransferases, with equal levels of expression ofthe two enzymes being achieved, a fermentation scale 14-21 times greater(or more) would be required for the ST6Gal I sialyltransferase relativeto the ST3Gal III sialyltransferase. For the ST6Gal I sialyltransferase,expression levels of 0.3 U/1 in yeast has been reported (Borsig et al.(1995) Biochem. Biophys. Res. Commun. 210: 14-20). Expression levels of1000 U/liter of the ST3 Gal III sialyltransferase have been achieved inAspergillus niger. At current levels of expression 300-450,000 liters ofyeast fermentation would be required to produce sufficient enzyme forsialylation of 1 kg of glycoprotein using the ST6Gal Isialyltransferase. In contrast, less than 10 liter fermentation ofAspergillus niger would be required for sialylation of 1 kg ofglycoprotein using the ST3Gal III sialyltransferase. Thus, thefermentation capacity required to produce the ST3Gal IIIsialyltransferase for a large scale sialylation reaction would be 10-100fold less than that required for producing the ST6Gal I; the cost ofproducing the sialyltransferase would be reduced proportionately.

Cri-IgG Antibody

8. Glyco-Remodeling of Cri-IgG1 Antibodies

This example sets forth the procedures for in vitro remodeling ofCri-IgG1 antibodies.

N-glycosylation at one conserved site at Asn 297 in the Fc domain of amonoclonal antibody can modulate its pharmacokinetic behavior andeffector functions (Dwek et al., 1995, J. Anat. 187:279-292; Boyd etal., 1995, Mol. Immunol. 32:1311-1318; Lund et al., 1995, FASEB J.1995,9:115-119; Lund et al., 1996,J. Immunol. 157:4963-4969; Wright &Morrison, 1998, J. Immunol. 160:3393-3402; Flynn & Byrd, 2000, Curr.Opin. Oncol. 12:574-581). During cell culture fermentation or in certainpathological conditions, significant heterogeneity arises in theglycosylation pattern at this site. The resulting different patterns ofglycosylation on the Fc domain are characterized by complex biantennarystructures with zero, one, and two terminal galactose residues (G0, G1,and G2, respectively, see Table 13). The observed glycoform variations,such as the variation in terminal galactosylation, truncatedN-glycoforms and bisecting modification, have been shown to influencethe antibody's therapeutic properties, especially its ability to mediatetargeted cell killing through complement binding and activation (Boyd etal., 1995, supra; Wright & Morrison, 1998, supra, Mimura et al., 2000,Molec. Immunol. 37:697-706; Davies et al., 2001, Biotechnol. Bioeng.74:288-294).

In order to obtain different glycoforms of Cri-IgG1 antibodies and testtheir Fc effector functions, Cri-IgG1 antibodies were trimmed backstepwise using exoglycosidases to generate glycoforms lacking sialicacid (G2, G1), glycoforms lacking sialic acid and galactose (G0), andglycoforms lacking sialic acid, galactose and N-acetyl glucos amine(M3N2F), as illustrated in Table 13. These molecules were subsequentlymodified using different glycosyltransferases and appropriate sugars.Modification conditions were developed that resulted in the conversionof the original antibody glycan structures into different glycoforms:M3N2, GnT-I-M3N2 (the M3M2 glycoform with a GlcNAc moiety added usingGnT-I), G0, Bisecting-G0 (the G0 moiety with a bisecting GlcNAc addedwith GnT-III), galactosylated bisecting-G0 (the bisecting-G0 glycoformwith terminal galactose moieties added), G2, mono-sialylated S1(α2,6)-G2(the G2 glycoform with one terminal sialic acid moiety added usingα2,6-sialyltransferase), S1(α2,3)-G2 (the G2 glycoform with one terminalsialic acid moiety added using α2,3-sialyltransferase) and disialylatedS2(α2,3)-G2 (the G2 glycoform). After every glycoremodeling step, theglycan structures were enzymatically released from the antibody proteinand were analyzed by various methods, including separation by capillaryelectrophoresis, 2-AA HPLC profiling and MALDI-TOF mass spectrometry.

TABLE 13 Abbreviations for glycoform structures. Abbreviation GlycanStructure(s) M3N2(F)

G0

G1

G2

The materials are methods used in these experiments are now described.

The Cri-IgG1 Monoclonal Antibody. The Cri-IgG1 antibody was obtainedfrom R. Jefferies, MRC Center for Immune Regulation, The Medical School,University of Birmingham, UK. The antibody is a non-recombinantantibody, and is isolated from a human myeloma. The antibody wasprepared using three methods. In the first method, referred to as“DEAE,” the antibody was isolated under relatively mild conditions usinga DEAE ion exchange column. In the second method, referred to as “SPA,”the antibody was purified on a protein A column (Staphylococcus aureusprotein A) with a low pH elution step. In the third method, referred toas “Fc,” the antibody was treated with a protease so that only the Fcportion of the antibody remained and the antigen binding domains wereremoved. These methods for antibody purification are well known to thoseof skill in the art and are not repeated in detail here.

Affinity purification of remodeled antibodies. Antibody, modified eitherby exoglycosidase or glycosyltransferase, was affinity purified on aProA-sepharose 4-fast flow column (Amersham Bioscience, ArlingtonHeights, Ill.), eluted with 0.1 M glycine-HCl buffer, pH 2.7, andimmediately neutralized with 1 M Tris, pH 9.5. The eluates werebuffer-exchanged using a NAP-10 column (Amersham Bioscience, ArlingtonHeights, Ill.) to an appropriate buffer for the next step ofglycosylation, such as 100 mM MES, pH 6.5 or 50 mM Tris-HCl, pH 7.2. Theremodeled final products were dialyzed extensively against PBS at 4° C.in Tube-O-Dialyzers™ (Chemicon International, Temecula, Calif.) with aMWCO of 8 kDa.

In vitro glycosidase treatment of Cri-antibodies. Antibody wasbuffer-exchanged into 50 mM Na phosphate/Citrate, pH 6.0 using NAP-10column (Amersham Bioscience, Arlington Heights, Ill.). In vitro trimmingback of sugar moieties was carried out stepwise, by contacting theantibody (5 mg/mL) with 20 mU/mg protein neuraminidase at 37° C.overnight (to remove terminal sialic acid moieties ), 20 mU/mg proteinβ-galactosidase at 37° C., overnight (to remove terminal galactosemoieties to result in the G0 glycoform), and/or 2 U/mgβ-N-acetylhexosaminidase (from Jack Bean, Seikagaku, Tokyo, Japan) at37° C., overnight (to remove terminal N-acetyl glucosamine to result inthe M3N2 glycoform). The samples were affinity purified as describedabove.

In vitro glycosylation of Cri-antibodies. In vitro GnT1 modification wasperformed using 1 mg/ml of the M3N2 glycoform antibody as the substrate,and 25 mU/mg of recombinant humanβ1,2-mannosyl-UDP-N-acetylglucosaminosyltransferase in a buffer of 100mM MES, pH 6.5, 5 mM MnCl₂, 5 mM UDP-GlcNAc, and 0.02% NaN₃ at 32° C.for 24 hr. An aliquot was removed for glycan analysis, and the resultingproducts were affinity purified as described above.

In vitro modification of the bisecting-glycoform was carried out using 1mg/ml of the M3N2 glycoform antibody as the substrate and 25 mU/mg ofβ1,2-recombinant human mannosyl-UDP-N-acetylglucosaminosyltransferase I,25 mU/mg of β1,2-recombinant humanmannosyl-UDP-N-acetylglucosaminosyltransferase II and 3.5 mU/mg ofβ1,4-recombinant mouse mannosyl-UDP-N-acetylglucosaminosyltransferaseIII in a buffer of 100 mM MES pH 6.5, 10 mM MnCl₂, 5 mM UDP-GlcNAc, and0.02% NaN₃ at 32° C. for 24 hrs. An aliquot was removed for glycananalysis, and the remaining product was affinity purified as describedabove.

In vitro galactosylation was performed using G0 glycoform antibody orbisecting glycoform antibody by contacting the antibody with 0.6 U/mgrecombinant bovine milk β1,4 galactosyltransferase in a buffer of 50 mMTris-HCl pH 7.4, 150 mM NaCl, 5 mM UDP-galactose, 5 mM MnCl₂, at 32° C.for 24 hrs. An aliquot was removed for glycan analysis, and theremaining products were affinity purified as described above.

In vitro sialylation was carried out using the G2 glycoform antibody (1mg/mL) by contacting it with 0.1 U/mg ST3Gal3 or 0.1 U/mg ST6Gall, 5 mMCMP-sialic acid, at 32° C. for 24 hr in a buffer of 50 mM Tris pH 7.4,150 mM NaCl, and 3 mM CMP-SA. An aliquot was removed for glycananalysis, and the remaining products were affinity purified as describedabove.

Glycan Analysis:

Capillary Electrophoresis with Laser Induced Fluorescence Dectection.Buffer components and nucleotide sugars were removed from an aliquot ofthe glycoremodeled antibody by dilution and concentration in a Microcon™YM-30 microconcentrator (Millipore, Bedford, Mass.). N-linkedoligosaccharides were released from the protein by contacting it withPNGase F (Prozyme, San Leandro, Calif.) using the methodology providedby the manufacturer. In brief, the sample was denatured in the buffer of50 mM sodium phosphate pH 7.5, 0.1% SDS, and 50 mM β-mercaptoethanol for10 min at 100° C. TX100 was then added to 0.75% (v/v) as well as 10UPNGaseF/200 μg protein. After 3 hours incubation at 37° C., the proteinwas ethanol precipitated and the supernatant was dried down. Thereleased free oligosaccharides were then labeled with8-aminopyrene-1,3,6-trisulfonic acid and analyzed by capillaryelectrophoresis with a carbohydrate labeling and analysis kit fromBeckman-Coulter, Inc. (Fullerton, Calif.), as indicated by themanufacturer (see also, Ma and Nashabeh, 1999, Anal. Chem.71:5185-5192).

Capillary electrophoresis (CE) was carried out in an eCAP™ N—CHO coatedCapillary (50 μm I.D., length to detector 40 cm; Beckman-Coulter, Inc.,Fullerton, Calif.), using a P/ACE™ MDQ Glycoprotein System(Beckman-Coulter, Inc. Fullerton, Calif.) with Laser InducedFluorescence Detector (Beckman-Coulter, Inc. Fullerton, Calif.). Sampleswere introduced into the cartridge by 20 psi pressure for 10 sec. andseparated under 25 kV with reverse polarity for 20 min. Cartridgetemperature was kept at 20° C. The electropherogram was generated bylaser-induced fluorescence detection at an excitation wavelength of 488nm and an emission wavelength of 520 nm.

Carbohydrate standards (Calbiochem®, EMD Biosciences, Inc., San Diego,Calif.), including M3N2 (N-linked trimannosyl core without core fucose),G0 (N-linked oligosaccharide, asialo, agalacto, biantennary with corefucose), G2 (N-linked oligosaccharide, asialo, biantennary with corefucose), and G2 without fucose, S1-G2 (mono-sialylated, galactosylatedbiantennary oligosaccharide without core fucose) and S2-G2(di-sialylated, galactosylated biantennary oligosaccharide without corefucose), (from Glyko, see, ProZyme, San Leandro, Calif.), M3N2F(N-linked trimannosyl core with core fucose) and NGA2F (N-linkedoligosaccharide asialo, agalacto, biantennary with core fucose and withbisecting GlcNAc) were labeled with 1-aminopyrene-3,6,8-trisulfonate(APTS, Beckman-Coulter, Inc. Fullerton, Calif.) and used to identify thedistribution of glycans released from the antibody.

2-AA HPLC. PNGaseF released glycans were labeled with 2-AA(2-anthranilic acid) according to the method described by Anumula andDhume with slight modifications (1998, Glycobiology 8:685-694):Reductively-aminated N-glycans were analyzed using a Shodex AsahipakNH2P-50 4D amino column (4.6 mm×150 mm) (Showa Denko K. K., Tokyo,Japan). The two solvents used for the separation are A) 2% acetic acidand 1% tetrahydrofuran in acetonitrile and B) 5% acetic acid, 3%triethylamine and 1% tetrahydrofuran in water.

To separate neutral 2AA-labeled glycans, the column was elutedisocratically with 70% A for 5 minutes, followed by a linear gradientover a period of 60 minutes going from 70% to 50% B, followed by a steepgradient over a period of 5 minutes going from 50% to 5% B and a finalisocratic elution with 5% B for 10 minutes. Eluted peaks were detectedusing fluorescence detection with an excitation at 230 nm and detectionwavelength at 420 nm. In this gradient condition, the G0 glycoform willelute at about 30.5 minutes, the G1 glycoform at about 34.0 minutes andthe G2 glycoform at about 37.0 minutes. Under these conditions, thepresence of fucose does not change the elution time.

To separate anionic 2AA-labeled glycans, the column was elutedisocratically with 70% A for 2.5 minutes, followed by a linear gradientover a period of 97.5 min going from 70% to 5% A and a final isocraticelution with 5% A for 15 minutes. Eluted peaks were detected usingfluorescence detection with excitation at 230 nm and detection at 420nm. In this gradient, neutral glycans are expected to elute between18.00-29.00 minutes, glycans with one charge elute between 30.00-40.00minutes, glycans with two charges elute between 43.00-52.00 minutes,glycans with three charges elute between 54.00-63.00 minutes, andglycans with four charges elute between 65.00-74.00 minutes.

MALDI analysis of reductively-aminated N-glycans. A small aliquot of thePNGase-released N-glycans that were labeled with 2-anthranilic acid(2AA) were then dialyzed for 45 minutes on a MF-Millipore membranefilter (0.025 μm pore, 47 mm dia.), which was floating on water. Thedialyzed aliquot was dried in a Speedvac™ (ThermoSavant, Holbrook,N.Y.), redissolved in a small amount of water, and mixed with a solutionof 2,5-dihydroxybenzoic acid (10 g/L) dissolved in water/acetonitrile(50:50).

The mixture was dried onto a MALDI target and analyzed using an AppliedBiosystems DE-Pro mass spectrometer (Applied Biosystems, Inc., FosterCity, Calif.) operated in the linear/negative-ion mode. Oligosaccharidestructures were assigned based on the observed mass-to-charge ratio andliterature precedence. No attempt was made to fully characterizeisobaric structures.

SDS-PAGE. To determine the stability of the glycoremodeled antibody, allthe samples were analyzed by SDS-PAGE. The final products of the sampleswere run under non-reducing conditions using 8-1 6% Tris-glycine gel(Invitrogen, Carlsbad, Calif.). Bovine serum albumin was run underreducing condition as quantitative standards. The gel was stained withGelCode Blue Stain Reagent (Pierce Chemical Co., Rockford, Ill.) forvisualization.

The results of the experiments are now described.

Native glycoforms of Cri expressed in human myeloma cells. Cri-IgG1antibody purified from the serum of a patient having multiple myelomacontains variable glycoforms. FIGS. 97A-97C shows the HPLC profiles ofglycans enzymatically released from Cri-IgG1 antibody. FIGS. 98A-98Cshows the MALDI profiles of glycans enzymatically released from Cri-IgG1antibody expressed in human myeloma cells. The major forms areunder-galactosylated G0, G1, while G2 and sialylated structures arerelatively minor (Table 14 and FIG. 97C). To test the impact of modifiedglycans on the therapeutic properties of the monoclonal antibody,Cri-IgG1 antibody was modified by performing in vitro exoglycosidasestrimming and in vitro glycosylation remodeling to generate differentglycoforms of this antibody.

TABLE 14 Relative amount of different glycoforms of human myelomacell-expressed Cri-IgG1 separated by HPLC was calculated from the areasof individual peaks. Criantibodies S1G2 G2 G1 G0 DEAE 45.04 54.96 SPA 63.17 48.25 51.75 Fc 51.41 38.83

Initially, optimization of each step in exoglycosidases trimming andglycosylation was performed at small scale (100 μg of each).

Trimannosyl core glycoform of Cri-IgG1 Antibody (M3N2). M3N2 was createdby stepwise treatment of glycosidases, including neuraminidase,β1,4-galactosidase and β1-2, 3, 4, 6 N-acetylhexosaminidase. To assessthe removal of terminal galactose and GlcNAc on the glycoremodeledCri-IgG1 antibody samples, a quantitative capillary electrophoresis (CE)method was used. The glycans were enzymatically released from theglycoremodeled antibody with PNGase F and were derivatized with8-aminopyrene-1,3,6-trisulfonic acid (APTS) at the reducing terminus.The resulting products were analyzed by CE with on-column laser-inducedfluorescence detection (LIF) (Ma & Nashabeh, 1999, supra). Since theseparation of the glycans is based on the differences in hydrodynamicsize, the APTS labeled glycans migrate in order of increasing size(M3N2<M3N2F<G0<G1<G2).

FIGS. 99A-99D show the electropherograms indicating the glycans releasedfrom glycoremodeled Cri-IgG1 antibody as well as glycan standardsderivatized with APTS (FIG. 99A). The glycoforms were identified bycomparing their electrophoretic mobilities to the standards. Therelative amount of each glycan species was calculated from the relativearea percentage of each indicated peak, and the results are presented inTable 15. The M3N2F glycoform represents 91% of the glycans of DEAE-Cri,80% of the glycans of SPA-Cri, and 100% of the glycans of Fc-Cri.Incomplete removal of GlcNAc moiety resulting in the GnT-I-M3N2Fglycoform (see, Table 15) was observed in the glycan structures fromDEAE-Cri (8.6%) and SPA-Cri (˜20%). Glycoform GnT-I-M3N2F is the M3N2Fglycoform with one additional GlcNAc, such as would be added by GnT-I.

TABLE 15 The areas of individual peaks from CE profile in FIG. 99 werecalculated, and relative amounts of the M3N2F and GnT-I-M3N2F glycoformswere determined. M3N2F GnT-I-M3N2F RT (min.) % RT (min.) % DEAE 10.13391.4 10.842 8.6 SPA 10.133 80.01 10.842 19.99 Fc 10.133 100 10.842 0

Degalactosylated glycoform (G0). Cri-IgG1 antibody with G0 glycoformswas obtained by stepwise treatment the native Cri-IgG1 antibody withneuraminidase and β1,4-galactosidase in for 24 hours for each reaction.The glycans released from the glycoremodeled antibody were analyzed byCE, HPLC and MALDI. FIG. 100A shows the CE profile of the releasedglycans. In all three samples, only one peak was observed which wasdesignated as the G0 glycoform based on comparison with the standards(FIG. 100A and Table 16).

TABLE 16 The relative amount of the G0 glycoform of Cri-IgG1 determinedby CE and HPLC. CE HPLC RT (min.) % RT (min.) % DEAE 11.408 100.0 31.194100.0 SPA 11.408 100.0 31.194 100.0 Fc 11.408 100.0 31.194 100.0

In addition to the glycan analysis provided by CE, a quantitative HPLCmethod was also used to determine the percent of the G0 glycoformrepresented by remodeled glycans of the Cri-IgG1 antibody. The glycandistribution on the glycoremodeled antibody was monitored byenzymatically releasing the glycans with PNGase F and derivatizing thereleased products with 2-anthranilic acid (2-AA) at the reducingterminus. The derivatized mixture was separated by HPLC on a ShodexAsahipak NH2P-50 4D column with fluorescence detection. FIGS. 101A-101Cshow the chromatograms obtained from the released glycans. HPLC resultsconfirmed CE analysis, as only one major peak was found in all threesamples. In agreement with CE and HPLC data, MALDI analysis also showedalmost complete glycoremodeling to the G0 glycoform (FIGS. 102A-102C).

Fully galactosylated G2 glycoform (G2). Cri-IgG antibodies were treatedwith neuraminidase to yield asialo-glycoforms which were also undergalactosylated. These asialoglycoforms were then treated with 0.6 U/mlof bovine β1,4 galactosyltransferase and a galactose donor molecule toglycoremodel the antibody to have the G2 glycoform.

The extent of terminal galactosylation was determined by glycananalysis. Only one major peak was observed in both CE and HPLC profiles(FIGS. 103A-103C and FIGS. 104A-104C). This peak corresponds to the G2glycoform in each case. Calculation of the percent total peak areashowed almost complete (˜90%) conversion to the G2 from the undergalactosylated glycoforms of the original samples (see, Table 14). Theseresults are summarized in Table 17. MALDI analysis of the glycansfurther supported the almost to complete glycoremodeling to the G2glycoform in all of the samples (FIGS. 105A-105C).

TABLE 17 Relative amount of G2 glycoform of remodeled Cri-IgI1 antibodydetermined by percent total peak area in CE and HPLC analysis. CE HPLCRT (min.) % RT (min.) % DEAE 12.94 90 31.194 100 SPA 12.94 92 31.194 90Fc 12.94 84 31.194 89

GnT-I-glycoform (GnT-I-M3N2). The M3N2 glycoform Cri-IgG antibody wasglycoremodeled to the GnT-I-M3N2 glycoform by adding one GlcNAc moietyto the molecule. The molecule was contacted with 25 mU GnT-I/mg antibodyand an appropriate GlcNAc donor molecule. CE, HPLC and MALDI analysis ofreleased glycans (FIGS. 106A-106D, FIGS. 107A-107C and FIGS. 108A-108C,respectively) indicated that the original M3N2F glycoform was completelyremodeled. However, only 40-60% of the modified structures were theGnT-I-M3N2 glycoform, and about 30% were the G0 glycoform. The presenceof the G0 glycoform may be the result of incomplete GlcNAc trimming whenmaking the original M3N2 form.

Bisecting glycoform (NGA2F). The M3N2 glycoform Cri-IgG antibody wasglycoremodeled to the NGA2F glycoform by contacting it with acombination the three transferases, GnT-I, GnT-II and GnT-III, and anappropriate N-acetylglucosamine donor molecule. The reaction wascompleted in 24 hours. To determine the extent to which thebisecting-GlcNAc moiety was added to the glycans, CE analysis was usedto determine the glycoforms present on the glycoremodeled antibody.

FIGS. 109A-109D shows the electropherograms obtained from CE analysis ofthe glycans released from glycoremodeled Cri-IgG1 antibody. Four peaksappeared after remodeling. A major peak migrated at the same retentiontime as the NGA2F standard glycoform. The three other minor peaks arelikely to be the incompletely remodeled glycans. For comparison, aquantitative HPLC method was also used, where the 2-AA labeled glycanseluted in order of increasing size (Gn1<G0<NGA2F). As shown in FIGS.110A-110C, similar results were obtained from the CE analysis of theglycans. No M3N2F was found using either the CE or HPLC analysis. NGA2Fglycans were the major peaks I both CE and BPLC analysis. The Gn1 and G0glycans still remaining in the sample likely are the result ofincomplete modification. Most of the original M3N2F glycoforms wereremodeled by three GlcNAc moieties to the NGA2F glycoform (60˜70%),about 15˜18% were remodeled by the addition of two GlcNAc moieties tothe G0 glycoform, and only small amount (˜7%) were remodeled by theaddition of only one GlcNAc moiety. MALDI-MS analysis of the releasedglycans (FIGS. 111A-111C) shows peaks of glycoforms with one, two orthree terminal GlcNAc moieties, in agreement with CE and HPLC analysis(FIGS. 109 and 110). The relative amount of each glycan species wascalculated from the relative area percentage of each indicated peak, andis summarized in Table 18.

TABLE 18 Relative amounts of different glycoforms from GnT-I, II, andIII remodeled Cri-IgG1, as determined by CE and HPLC. Retention % PeakArea (min.) DEAE SPA Fc CE Peak 1 10.238 6.39 6.89 7.98 Peak 2 10.77515.82 14.29 17.9 Peak 3 11.325 14.14 8.87 15.69 Bisec. 11.625 63.6570.04 58.43 HPLC Peak 1 21.117 37.4 15.02 14 Peak 2 26.817 12.9 14.2410.15 Peak 3 31.224 14.78 2.11 30.2 Bisec. 32.078 34.93 68.63 45.64

Galactosylated Bisecting (Gal-NGA2F glycoforms. NGA2F glycoforms ofCri-IgG1 antibodies were glycoremodeled with bovineβ1,4-galactosyltransferase and an appropriate galactose donor. Theterminal galactose moieties were added using 0.6 U/ml of β1,4galactosyltransferase. FIGS. 112A-12D shows the electropherogramsobtained using the 2-AA HPLC method. In brief, the glycoformsterminating in GalNAc were almost 100% galactosylated. Comparing FIG.112A to FIG. 112B for DEAE Cri-IgG1, and FIG. 112C to FIG. 112D for FcCri-IgG 1, the 2-AA HPLC profile of GnT-I, II and III modified glycans(FIGS. 112A and 112C) is modified by GalT1 so that all of the glycanpeakes were shifted to elute later due to the size increase from addedgalactose moieties (FIGS. 112B and 112D). These results were furtherconfirmed by MALDI-MS analysis.

Sialylated (S2G2) glycoforms of Cri-IgG1. The glycoremodeled G2glycoforms of Cri-IgG1 antibody were further remodeled using bothST3Gal3 and ST6Gal1. FIGS. 113A-113C shows the HPLC profile of the G2glycoforms remodeled with ST3Gal3. Most of the G2 glycoforms wereconverted into S2G2 glycoforms (the G2 glycoform with 2 additionalterminal sialic acid moieties; ˜70%, see, Table 19), and only smallamounts were the S1G2 glycoform (the G2 glycoform with 1 additionalterminal sialic acid moiety; <25%, see Table 19). These results werefurther confirmed in the MALDI analysis shown in FIGS. 114A-114C. MALDIdata also shows that all the G2 glycoforms were sialylated to eitherS2G2 or S1G2 glycoforms.

TABLE 19 Relative amounts of different glycoforms from ST3Gal3 remodeledCri-IgG1 as determined by HPLC. RT (min.) DEAE SPA Fc S1G2 36.7 25.624.83 23.39 S2G2 46.9 4.12 6.83 49.4 58.93 50.68 61.88 52.19 9.1 7.566.07

By comparison, ST6Gal1 remodeling of the G0 glycoform did not reach thelevel of completion found with ST3Gal3 remodeling. FIGS. 115A-115D andFIGS. 116A-116C show the results obtained from CE and HPLC analysis,respectively. No S2G2 glycoforms were seen in any of the glycoremodeledsamples. However, all of the G2 glycoforms were converted into S1-G2.Analysis from MALDI-MS also supports these data (FIGS. 117A-117C).

Stability of remodeled glycans of Cri-IgG1. Lastly, the stability of theCri-IgG1 glycans remodeled by exoglycosidase treatment and glycosylationwas investigated. Each glycoremodeled Cri-IgG1 antibody was stored at 4°C., and was checked by SDS-PAGE for degradation at two weeks afterremodeling. As shown in FIGS. 118A-118E, the remodeled DEAE and SPAantibodies both retained a molecular weight of about 150 kDa, indicatinglittle to no degradation, regardless of the kind of glycoremodelingperformed. The Fc Cri-IgG1 antibody retained a molecular weight of about38 kDa, also indicating little to no degradation, regardless of the kindof remodeling performed.

Effector Function Bioassay of Remodeled Cri-IgG1 antibodies. Theeffector function bioassay was derived from the procedure of Mimura etal. (2000, Molecular Immunology 37:697-706). The IC₅₀ of the glycoformsof Cri-IgG1 antibody was determined by inhibition of the superoxideresponse of U937 cells elicited by red blood cells sensitized withnative anti-NIP antibody.

Monocytic U937 cells were cultured in the presence of 1000 units/mLinterferon gamma for 2 days to induce the differentiation of the cellsand their capacity to generate superoxide. The cells were then washedand resuspended at 2×10⁶ cells/mL in Hanks balanced salt solutionwithout phenol red and containing 20 mM HEPES pH 7.4 and 0.15 mM BSA.The red blood cells were sensitized with anti-NIP(5-iodo4-hydroxy-3-nitrophenacetyl) antibody, in the absence or presenceof the various glycoforms of Cri-IgG1 antibody, with incubation at 37°C. for 30 minutes. The cells were then washed three times with PBS andresuspended at 2.5×10⁷ cells/mL in HBSS-BSA. The U937 cells (100 μl,2×10⁶ cells/mL) were added to plastic tubes and lucigenin (20 μl, 2.5mM) was added to the tubes. The tubes were warmed in a 37° C. water bathfor 5 minutes. The sensitized red blood cells (80 μl, 2.5×10⁷ /mL) werethen added to the tubes. Superoxide anion production was measured bylucigenin-enhanced chemiluminescence at 37° C. over a 30 minute periodusing a Berthold LV953 luminometer (Berthold Australia Pty Ltd,Bundoora, Australia).

The G0 and M3N2 glycoforms Cri-IgG1 antibody had relative inhibitoryvalues of 92% and 85%, respectively, as compared with the nativeantibody. However, the native CRI-IgG1 antibody lacked core fucose.Shields et al. (2002, J. Biol. Chem. 277:26733-26740) suggests that thelack of core fucose will improve inhibitory values 10 fold. Based onthese results, it is anticipated that inhibitory values of thegalactosylated-bisecting-G0 glycoform will be greater than thebisecting-G0 glycoform, which in turn will be much greater than the G2glycoform, which in turn will be approximately equal to thedisialylated-G2 glycoform and the monosialylated-G2 glycoform, which inturn will be greater than the native antibody glycoform, which in turnwill be greater than the G0 glycoform, which in turn will be greaterthan the M3N2 glycoform.

Complement Receptor-1

9. Sialylation and Fucosylation of TP10

This example sets forth the preparation of TP10 with sialyl Lewis Xmoieties and analysis of enhanced biological activity.

Interrupting blood flow to the brain, even for a short time, can triggerinflammatory events within the cerebral microvasculature that canexacerbrate cerebral tissue damage. The tissue damage that accrues isamplified by activation of both inflammation and coagulation cascades.In a murine model of stroke, increased expression of P-selectin andICAM-1 promotes leukocyte recruitment. sCR1 is recombinant form of theextracellular domain of Complement Receptor-1 (CR-1). sCR-1 is a potentinhibitor of complement activation. sCR1sLe^(X) (CD20) is an alternatelyglycosylated form of sCR1 that is alternately glycosylated to displaysialylated Lewis^(X) antigen. Previously, sCR-1sLeX that was expressedand glycosylated in vivo in engineered Lec11 CHO cells was found tocorrectly localize to ischemic cerebral microvessels and C1 q-expressingneurons, thus inhibiting neutrophil and platelet accumulation andreducing cerebral infarct volumes (Huang et al., 1999, Science285:595-599). In the present example, sCR1sLe^(X) which was prepared invitro by remodeling of glycans, exhibited enhanced biological activitysimilar to that of sCRsLe^(X) glycosylated in vivo.

The TP10 peptide was expressed in DUK B 11 CHO cells. This CHO cell lineproduces the TP10 peptide with the typical CHO cell glycosylation, withmany but not all glycans capped with sialic acid.

Sialylation of 66 mg of TP10. TP10 (2.5 mg/mL), CMPSA (5 mM), andST3Gal3 (0.1 U/mL) were incubated at 32° C. in 50 mM Tris, 0.15M NaCl,0.05% sodium azide, pH 7.2 for 48 hours. Radiolabelled CMP sialic acidwas added to a small aliquot to monitor incorporation. TP10 wasseparated from nucleotide sugar by SEC HPLC. Samples analyzed at 24hours and 48 hours demonstrated that the reaction was completed after 24hours. The reaction mixture was then frozen. The reaction products weresubjected to Fluorophore Assisted Carbohydrate Electrophoresis (FACE®;Glyko, Inc, Novato Calif.) analysis (FIG. 119).

Pharmacokinetic studies. Rats were purchased with a jugular veincannula. 10 mg/kg of either the pre-sialylation or post-sialylation TP10peptide was given by tail vein injection to three rats for eachtreatment (n=3). Fourteen blood samples were taken from 0 to 50 hours.The concentration in the blood of post-sialylation TP10 peptide washigher than that of pre-sialylation TP10 at every time point past 0 hour(FIG. 120). Sialic acid addition doubled the area under the plasmaconcentration-time curve (AUC) of the pharmacokinetic curve as comparedto the starting material (FIG. 121).

Fucosylation of sialylated TP10. 10 mL (25 mg TP10) of the abovesialylation mix was thawed, and GDP-fucose was added to 5 mM, MnCl₂ to 5mM, and FTVI (fucosyltransferase VI) to 0.05 U/mL. The reaction wasincubated at 32° C. for 48 hours. The reaction products were subjectedto Fluorophore Assisted Carbohydrate Electrophoresis (FACE®; Glyko, Inc,Novato Calif.) analysis (FIG. 122). To a small aliquot, radiolabelledGDP-fucose was added to monitor incorporation. TP10 was separated fromnucleotide sugar by SEC HPLC. Samples analyzed at 24 hours and 48 hoursdemonstrated that the reaction was completed at 24 hours. An in vitroassay measuring binding to E-selectin indicate that fucose addition canproduce a biologically-active E-selectin ligand (FIG. 123).

Enbrel™

10. GlycoPEGylation of an Antibody Enbrel™

This example sets forth the procedures to PEGylate the O-linked glycansof an antibody molecule. Here, Enbrel™ is used as an example, howeverone of skill in the art will appreciate that this procedure can be usedwith many antibody molecules.

Preparation of Enbrel™-SA-PEG (10 kDa). Enbrel™(TNF-receptor-IgG₁-chimera), either with the O-linked glycans sialylatedprior to PEGylation or not, is dissolved at 2.5 mg/mL in 50 mM Tris-HCl,0.15 M NaCl, 5 mM MnCl₂, 0.05% NaN₃, pH 7.2. The solution is incubatedwith 5 mM UDP-galactose and 0.1 U/mL of galactosyltransferase at 32° C.for 2 days to cap the undergalactosylated glycans with galactose. Tomonitor the incorporation of galactose, a small aliquot of the reactionhas ¹⁴C-galactose-UDP ligand added; the label incorporated into thepeptide is separated from the free label by gel filtration on a TosoHaas G2000SW analytical column in methanol and water. The radioactivelabel incorporation into the peptide is quantitated using an in-lineradiation detector.

When the reaction is complete, the solution is incubated with 1 mMCMP-sialic acid-linker-PEG (10 kDa) and 0.1 U/mL of ST3Gal3 at 32° C.for 2 days. To monitor the incorporation of sialic acid-linker-PEG, thepeptide is separated by gel filtration on a Toso Haas G3000SW analyticalcolumn using PBS buffer (pH 7.1). When the reaction is complete, thereaction mixture is purified using a Toso Haas TSK-Gel-3000 preparativecolumn using PBS buffer (pH 7.1) and collecting fractions based on UVabsorption. The fractions containing product are combined, concentrated,buffer exchanged and then freeze-dried. The product of the reaction isanalyzed using SDS-PAGE and IEF analysis according to the procedures andreagents supplied by Invitrogen. Samples are dialyzed against water andanalyzed by MALDI-TOF MS.

Erythropoietin (EPO)

11. Addition of GlcNAc to EPO

This example sets forth the addition of a GlcNAc residue on to atri-mannosyl core.

Addition of GlcNAc to EPO. EPO was expressed in SF-9 insect cells andpurified (Protein Sciences, Meriden, Conn.). A 100% conversion from thetri-mannosyl glycoform of Epo to the “tri-mannosyl core+2 GlcNAc” (Peak1, P1 in FIG. 124) was achieved in 24 hours of incubation at 32° C. with100 mU/ml of GlcNAcT-I and 100 mU/ml of GlcNAcT-II in the followingreaction final concentrations:

-   -   100 mM MES pH 6.5, or 100 mM Tris pH 7.5    -   5 mM UDP-GlcNAc    -   20 mM MnCl₂    -   100 mU/ml GlcNAcT-I    -   100 mU/ml GlcNAcT-II    -   1 mg/ml EPO (purified, expressed in Sf9 cells, purchased from        Protein Sciences).

Analysis of glycoforms. This assay is a slight modification on K-RAnumula and ST Dhume, Glycobiology 8 (1998) 685-69. N-glycanase (PNGase)released N-glycans were reductively labeled with anthranilic acid. Thereductively-aminated N-glycans were injected onto a Shodex AsahipakNH2P-50 4D amino column (4.6 mm×150 mm). Two solvents were used for theseparation: A) 5% (v/v) acetic acid, 1% tetrahydrofuran, and 3%triethylamine in water, and B) 2% acetic acid and 1% tetrahydrofuran inacetonitrile. The column was then eluted isocratically with 70% B for2.5 minutes, followed.by a linear gradient over a period of 97.5 minutesgoing from 70 to 5% B and a final isocratic elution with 5% B for 15minutes. Eluted peaks were detected using fluorescence detection with anexcitation of 230 nm and emission wavelength of 420 nm.

Under these conditions, the trimannosyl core had a retention time of22.3 minutes, and the product of the GnT reaction has a retention timeof 30 minutes. The starting material was exclusively trimannosyl corewith core GlcNAc (FIG. 124).

12. Preparation of EPO with Multi-Antennary Complex Glycans

This example sets forth the preparation of PEGylated, biantennary EPO,and triantennary, sialylated EPO from insect cell expressed EPO.

Recombinant human erythropoietin (rhEPO) from the baculovirus/Sf9expression system (Protein Sciences Corp., Meriden, Conn.) was subjectedto glycan analysis and the resulting glycans were shown to be primarilytrimannosyl core with core fucose, with a small percentage of glycansalso having a single GlcNAc.

Addition of N-acetylglucosamine with GnT-I and GnT-II. Two lots of rhEPO(1 mg/mL) were incubated with GnT-I and GnT-II, 5 mM UDP-glcNAc, 20 mMMnCl₂, and 0.02% sodium azide in 100 mM MES pH 6.5 at 32° C. for 24 hr.Lot A contained 20 mg of EPO, and 100 mU/mL GnT-I and 60 mU/mL GnT-II.Lot B contained 41 mg of EPO, and 41 mU/mL GnT-I+50 mU/mL GnT-II. Afterthe reaction, the sample was desalted by gel filtration (PD10 columns,Pharmacia LKB Biotechnology Inc., Piscataway, N.J.).

EPO glycans analyzed by 2-AA HPLC profiling. This assay is a slightmodification on Anumula and Dhume, Glycobiology 8 (1998) 685-69.Reductively-aminated N-glycans were injected onto a Shodex AsahipakNH2P-50 4D amino column (4.6 mm×150 mm). Two solvents were used for theseparation, A) 5% (v/v) acetic acid, 1% tetrahydrofuran, and 3%triethylamine in water and B) 2% acetic acid and 1% tetrahydrofuran inacetonitrile. The column was then eluted isocratically with 70% B for2.5 min, followed by a linear gradient over a period of 100 min goingfrom 70 to 5% B, and a final isocratic elution with 5% B for 20 min.Eluted peaks were detected using fluorescence detection with anexcitation of 230 nm and emission wavelength of 420 nm. Non-sialylatedN-linked glycans fall in the LC range of 23-34 min, monosialylated from34-42 min, disialylated from 42-52 min, trisialylated from 55-65 min andtetrasialylated from 68-78 min.

Glycan profiling by 2AA HPLC revealed that lot A was 92% converted to abiantennary structure with two GlcNAcs (the balance having a singleGlcNAc. Lot B showed 97% conversion to the desired product (FIGS. 125Aand 125B ).

Introducing a third antennary branch with GnT-V. EPO (1 mg/mL of lot B)from the product of the GnT-I and GnT-II reactions, after desalting onPD-10 columns and subsequent concentration, was incubated with 10 mU/mLGnT-V and 5 mM UDP-GlcNAc in 100 mM MES pH 6.5 containing 5 mM MnCl₂ and0.02% sodium azide at 32° C. for 24 hrs. 2AA HPLC analysis demonstratedthat the conversion occurred with 92% efficiency (FIG. 126).

After desalting (PD-10) and concentration, galactose was added withrGalTI: EPO (1 mg/mL) was incubated with 0.1 U/mL GalT, 5 mMUDP-galactose, 5 mM MnCl₂ at 32° C. for 24 hrs.

MALDI analysis of reductively-aminated N-glycans from EPO. A smallaliquot of the PNGase released N-glycans from EPO that had beenreductively labeled with anthranilic acid was dialyzed for 45 min on anMF-Millipore membrane filter (0.025 μm pore, 47 mm dia), which wasfloating on water. The dialyzed aliquot was dried in a speedvac,redissolved in a small amount of water, and mixed with a solution of2,5-dihydroxybenzoic acid (10 g/L) dissolved in water/acetonitrile(50:50). The mixture was dried onto the target and analyzed using anApplied Biosystems DE-Pro MALDI-TOF mass spectrometer operated in thelinear/negative-ion mode. Oligosaccharides were assigned based on theobserved mass-to-charge ratio and literature precedence.

Analysis of released glycans by MALDI showed that galactose was addedquantitatively to all available sites (FIG. 127). Galactosylated EPOfrom above was then purified by gel filtration on a Superdex 1.6/60column in 50 mM Tris, 0.15M NaCl, pH 6.

Sialylation. After concentration and desalting (PD-10), 10 mggalactosylated EPO (1 mg/mL) was incubated with ST3Gal3 (0.05 U/mL), andCMP-SA (3 mM) in 50 mM Tris, 150 mM NaCl, pH 7.2 containing 0.02% sodiumazide. A separate aliquot contained radiolabelled CMP-SA. The resultingincorporated label and free label was separated by isocratic sizeexclusion chromatography/HPLC at 0.5mL/min in 45% MeOH, 0.1% TFA(7.8mm×30 cm column, particle size 5 μm, TSK G2000SW_(XL), Toso Haas,Ansys Technologies, Lake Forest, Calif.). Using this procedure, 12% ofthe counts were incorporated (360 micromolar, at 33 micromolar EPO, orabout 10.9 moles/mole). Theoretical (3 N-linked sites, tri-antennary) isabout 9 moles/mole incorporation. These correspond within the limits ofthe method. In an identical reaction with ST6Gal1 instead of ST3Gal3,5.7% of the radiolabel was incorporated into the galactosylated EPO, orabout 48% compared with ST3Gal3.

13. GlycoPEGylation of EPO Produced in Insect Cells

This example sets forth the prepartion of PEGylated biantennary EPO frominsect cell expressed EPO.

Recombinant human erythropoietin (rhEPO) from the baculovirus/Sf9expression system (Protein Sciences Corp., Meriden, Conn.) was subjectedto glycan analysis and the resulting glycans were shown to be primarilytrimannosyl core with core fucose, with a small percentage of glycansalso having a single GlcNAc (FIG. 128).

Addition of N-acetylglucosamine with GnT-I and GnT-II. Two lots of rhEPO(1 mg/mL) were incubated with GnT-I and GnT-II, 5 mM UDP-glcNAc, 20 mMMnCl₂, and 0.02% sodium azide in 100 mM MES pH 6.5 at 32° C. for 24hr.Lot A contained 20 mg of EPO, and 100 mU/mL GnT-I and 60 mU/mL GnT-II.Lot B contained 41 mg of EPO, and 41 mU/mL GNT-I+50 mU/mL GnT-II. Afterthe reaction, the sample was desalted by gel filtration (PD10 columns,Pharmacia LKB Biotechnology Inc., Piscataway, N.J.).

Glycan profiling by 2AA HPLC revealed that lot A was 92% converted to abiantennary structure with two GlcNAcs (the balance having a singleglcNAc. Lot B showed 97% conversion to the desired product (FIGS. 125Aand 125B ).

Galactosylation of EPO lot A. EPO (˜16 mgs of lot A) was treated withGnT-II to complete the addition of GlcNAc. The reaction was carried outin 50 mM Tris pH 7.2 containing 150 mM NaCl, EPO mg/ml, 1 mM UDP-GlcNAc,5 mM MnCl₂, 0.02% sodium azide and 0.02 U/ml GnT-II at 32 C for 4 hrs.Then galactosylation of EPO was done by adding UDP-galactose to 3 mM andGalT1 to 0.5 U/ml and the incubation continued at 32° C. for 48 hrs.

Galactosylated EPO was then purified by gel filtration on a Superdex751.6/60 column in 50 mM Tris, 0.15M NaCl, pH 6. The EPO containing peakwas then analyzed by 2AA HPLC. Based on the HPLC data ˜85% of theglycans contains two galactose and ˜15% of the glycans did not have anygalactose after galactosylation reaction.

Sialylation of galactosylated EPO. Sialylation of galactosylated EPO wascarried out in 100 mM Tris pH containing 150 mM NaCl, 0.5 mg/ml EPO, 200mU/ml of ST3Gal3 and either 0.5 mM CMP-SA or CMP-SA-PEG (1 kDa) orCMP-SA-PEG (10 kDa) for 48 hrs at 32° C. Almost all of the glycans thathave two galactose residues were fully sialylated (2 sialicacids/glycan) after sialylation reaction with CMP-SA. MALDI-TOF analysisconfirmed the HPLC data.

PEGylation of galactosylated EPO. For PEGylation reactions usingCMP-SA-PEG (1 kDa) and CMP-SA-PEG (10 kDa), an aliquot of the reactionmixture was analyzed by SDS-PAGE (FIG. 129). The molecular weight of theEPO peptide increased with the addition of each sugar, and increasedmore dramatically in molecular weight after the PEGylation reactions.

In vitro bioassay of EPO. In vitro EPO bioassay (adapted from Hammerlinget al, 1996, J. Pharm. Biomed. Anal. 14: 1455-1469) is based on theresponsiveness of the TF-1 cell line to multiple levels of EPO. TF-1cells provide a good system for investigating the proliferation anddifferentiation of myeloid progenitor cells. This cell line wasestablished by T. Kitamura et al. in October 1987 from a heparinizedbone marrow aspiration sample from a 35 year old Japanese male withsevere pancytopenia. These cells are completely dependent on Interleukin3 or Granulocyte-macrophage colony-stimulating factor (GM-CSF).

The TF-1 cell line (ATCC, Cat. No. CRL-2003) was grown in RPMI+FBS10%+GM-CSF (12 ng/ml) and incubated at 37° C. 5% CO₂. The cells were insuspension at a concentration of 5000 cells/ml of media, and 200 μl weredispensed in a 96 well plate. The cells were incubated with variousconcentrations of EPO (0.1 μg/ml to 10 μg/ml) for 48 hours. A MTTViability Assay was then done by adding 25 μl of MTT at 5 mg/ml (SIGMAM5655), incubating the plate at 37° C. for 20 min to 4 hours, adding 100μl of isopropanol/HCl solution (100 ml isopropanol+333 μl HCl 6N),reading the OD at 570 nm, and 630 nm or 690 nm, and subtracting thereadings at 630 nm or 690 nm from the readings at 570 nm.

FIG. 130 contains the results when sialylated EPO, and EPOglycoPEGylated with 1 kDa or 10 kDa PEG was subjected to an in vitro EPObioactivity test. The EPO glycoPEGylated with 1 kDa PEG had almost thesame activity as the unglycoPEGylated EPO when both were at aconcentration of approximately 5 μg/ml. The EPO glycoPEGylated with 10kDa PEG had approximately half the activity of the unglycoPEGylated EPOwhen both were at a concentration of approximately 5 μg/ml.

14. GlycoPEGylation of O-Linked Glycans of EPO Produced in CHO Cells

Preparation of O-linked EPO-SA-PEG (10 kDa). Asialo-EPO, originallyproduced in CHO cells, is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15M NaCl, 0.05% NaN₃, pH 7.2. The solution is incubated with 5 mM CMP-SAand 0.1 U/mL of ST3Gal3 at 32° C. for 2 days. To monitor theincorporation of sialic acid onto the N-linked glycans, a small aliquotof the reaction had CMP-SA-¹⁴C added; the peptide is separated by gelfiltration on a Toso Haas G2000SW analytical column using methanol,water and the product detected using a radiation detector. When thereaction is complete, the solution is concentrated using a Centricon-20filter. The remaining solution is buffer exchanged with 0.05 M Tris (pH7.2), 0.15 M NaCl, 0.05% NaN₃ to a final volume of 7.2 mL until theCMP-SA could no longer be detected. The retentate is then resuspended in0.05 M Tris (pH 7.2), 0.15 M NaCl, 0.05% NaN₃ at 2.5 mg/mL protein. Thesolution is incubated with 1 mM CMP-SA-PEG (10 kDa) and ST3Gal1, toglycosylate the O-linked site, at 32° C. for 2 days. To monitor theincorporation of sialic acid-PEG, a small aliquot of the reaction isseparated by gel filtration suing a Toso Haas TSK-gel-3000 analyticalcolumn eluting with PBS pH 7.0 and analyzing by UV detection. When thereaction is complete, the reaction mixture is purified using a Toso HaasTSK-gel-3000 preparative column using PBS buffer (pH 7.0) collectingfractions based on UV absorption. The product of the reaction isanalyzed using SDS-PAGE and IEF analysis according to the procedures andreagents supplied by Invitrogen. Samples are dialyzed against water andanalyzed by MALDI-TOF MS.

15. EPO-Transferrin

This example sets forth the procedures for the glycoconjugation ofproteins to O-linked glycans, and in particular, transferrin isglycoconjugated to EPO. The sialic acid residue is removed from O-linkedglycan of EPO, and EPO-SA-linker-SA-CMP is prepared.EPO-SA-linker-SA-CMP is glycoconjugated to asialotransferrin withST3Gal3.

Preparation of O-linked asialo-EPO. EPO (erythropoietin) produced in CHOcells is dissolved at 2.5 mg/mL in 50 mM Tris 50 mM Tris-HCl pH 7.4,0.15 M NaCl, and is incubated with 300 mU/mL sialidase (Vibriocholera)-agarose conjugate for 16 hours at 32° C. To monitor thereaction a small aliquot of the reaction is diluted with the appropriatebuffer and a IEF gel performed according to Invitrogen procedures. Themixture is centrifuged at 10,000 rpm and the supernatant is collected.The supernatant is concentrated to a EPO concentration of about 2.5mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH 7.2. The solutionis incubated with 5 mM CMP-sialic acid and 0.1 U/mL of ST3Gal3 at 32° C.for 2 days. To monitor the incorporation of sialic acid, a small aliquotof the reaction had CMP-SA-fluorescent ligand added; the labelincorporated into the peptide is separated from the free label by gelfiltration on a Toso Haas G3000SW analytical column using PBS buffer (pH7.1). When the reaction is complete, the reaction mixture is purifiedusing a Toso Haas G3000SW preparative column using PBS buffer (pH 7.1)and collecting fractions based on UV absorption. The product of thereaction is analyzed using SDS-PAGE and IEF analysis according to theprocedures and reagents supplied by Invitrogen. Samples are dialyzedagainst water and analyzed by MALDI-TOF MS.

Preparation of EPO-SA-linker-SA-CMP. The O-linked asialo-EPO 2.5 mg/mLin 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH 7.2. The solution isincubated with 1 mM CMP-sialic acid-linker-SA-CMP and 0.1 U/mL ofST3Gal1 at 32° C. for 2 days. To monitor the incorporation of sialicacid-linker-SA-CMP, the peptide is separated by gel filtration on a TosoHaas G3000SW analytical column using PBS buffer (pH 7.1).

After 2 days, the reaction mixture is purified using a Toso Haas G3000SWpreparative column using PBS buffer (pH 7.1) and collecting fractionsbased on UV absorption. The product of the reaction is analyzed usingSDS-PAGE and IEF analysis according to the procedures and reagentssupplied by Invitrogen. Samples are dialyzed against water and analyzedby MALDI-TOF MS.

Preparation of Transferrin-SA-Linker-SA-EPO. EPO-SA-Linker-SA-CMP fromabove is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05%NaN₃, pH 7.2. The solution is incubated with 2.5 mg/mLasialo-transferrin and 0.1 U/mL of ST3Gal3 at 32° C. for 2 days. Tomonitor the incorporation of transferrin, the peptide is separated bygel filtration on a Toso Haas G3000SW analytical column using PBS buffer(pH 7.1) and the product detected by UV absorption. When the reaction iscomplete, the solution is incubated with 5 mM CMP-SA and 0.1 U/mL ofST3Gal3 (to cap any unreacted transferrin glycans) at 32° C. for 2 days.The reaction mixture is purified using a Toso Haas G3000SW preparativecolumn using PBS buffer (pH 7.1) collecting fractions based on UVabsorption. The product of the reaction is analyzed using SDS-PAGE andIEF analysis according to the procedures and reagents supplied byInvitrogen. Samples are dialyzed against water and analyzed by MALDI-TOFMS.

16. EPO-GDNF

This example sets forth the procedures for the glycoconjugation ofproteins, and in particular, the preparation of EPO-SA-Linker-SA-GDNF.

Preparation of EPO-SA-Linker-SA-GDNF. EPO-SA-Linker-SA-CMP from above isdissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH7.2. The solution is incubated with 2.5 mg/mL GDNF (produced in NSO) and0.1 U/mL of ST3Gal3 at 32° C. for 2 days. To monitor the incorporationof GDNF, the peptide is separated by gel filtration on a Toso HaasG3000SW analytical column using PBS buffer (pH 7.1) and the productdetected by UV absorption. When the reaction is complete, the solutionis incubated with 5 mM CMP-SA and 0.1 U/mL of ST3Gal3 (to cap anyunreacted GDNF glycans) at 32° C. for 2 days. The reaction mixture ispurified using a Toso Haas G3000SW preparative column using PBS buffer(pH 7.1) collecting fractions based on UV absorption. The product of thereaction is analyzed using SDS-PAGE and IEF analysis according to theprocedures and reagents supplied by Invitrogen. Samples are dialyzedagainst water and analyzed by MALDI-TOF MS.

17. Mono-Antennary GlycoPEGylation of EPO

This example sets forth the procedure for the preparation ofglycoPEGylated mono-antennary erythropoietin (EPO), and its bioactivityin vitro and in vivo.

When EPO (GenBank Accession No. P01588) is expressed in CHO cells,N-linked glycans are formed at amino acid residues 24, 38 and 83, and anO-linked glycan is formed at amino acid residue 126 (FIG. 131; Lai etal., 1986, J. Biol. Chem. 261:3116-3121). The bioactivity of thisglycoprotein is directly correlated with the level of NeuAc content.Increased sialic acid decreases the binding of EPO to its receptor invitro; however increased sialic acid increases the bioactivity of EPO invivo. The O-linked glycan has no impact on the in vitro or in vivoactivity of EPO, or the pharmacokinetics of the molecule (Wasley et al.,1991, Blood 77:2624-2632).

When EPO is expressed in insect cells, such as is accomplished using abaculovirus/Sf9 expression system (see also, Wojchowshi et al., 1987,Biochem. Biophys. Acta 910:224-232; Quelle et al., 1989, Blood74:652-657), N-linked glycans are formed at amino acid residues 24, 38and 83, but an O-linked glycan is not formed at amino acid residue 126(FIG. 132). This is because the insect cell does not have a glycosyltransferase that recognizes the amino acid sequence around amino acidresidue 126 of EPO. The majority of the N-linked glycans are composed ofGlcNAc₂Man₃Fuc. In the present example, EPO expressed in insect cellswas remodeled with high efficiency to achieve the complex glycanSA₂Gal₂GlcNAc₂Man₃FucGlcNAC₂ by contacting the protein with, in series,GnT1,2, GalT-1, and ST in the presence of the appropriate donormolecules. These enzymatic reactions were performed on insect cellexpressed EPO using reaction conditions disclosed herein, to yield thecomplex glycans herein with 92% total efficiency (Table 21). Optionally,O-linked glycans can also be added (O'Connell and Tabak, 1993, J. Dent.Res. 72:1554-1558; Wang et al., 1993, J. Biol. Chem. 268:22979-22983).

TABLE 21 Percent of each species of glycan structure in the populationof glycan structures on insect cell expressed EPO (“tsrating material”),and no EPO after each sequential enzymatic remodeling step. StartingGlycan Material After GnT-I, II After GalT-I After ST

 0.5%

98.0%

 1.0%  0.5%  0.5%

 0.5% 99.5%  4%  2%

95.9%  5%

92.0%

Also in the present example, EPO expressed in insect cells was remodeledto form mono-antennary, bi-anntenary and tri-antennary glycans, whichwere subsequently glycoPEGylated with 1 kDa, 10 kDa and 20 kDa PEGmolecules suing procedures described elsewhere herein. The molecularweights of these EPO forms were determined, and were compared toEpoetin™ having 3 N-linked glycans, and NESP (Aranesp™) having 5N-linked glycans (FIG. 133). Examples of the preparation of bi- andtri-antennary glycan structures are given in Example 7, herein.

EPO having monoantennary PEGylated glycan structures is prepared byexpressing EPO peptide in insect cells, then contacting the EPO peptidewith GnTI only (or alternatively GnTII only) in the presence of a GlcNAcdonor. The EPO peptide is then contacted with GalT-I in the presence ofa galactose donor. The EPO peptide is then contacted with ST in thepresence of SA-PEG donor molecules (FIG. 134A) to generate an EPOpeptide having three N-linked mono-antennary PEGylated glycan structures(FIG. 134B).

The in vitro bioactivity of EPO-SA and EPO-SA-PEG generated from insectcell expressed EPO was accessed by measuring the ability of the moleculeto stimulate the proliferation of TF-1 erythroleukemia cells.Tri-antennary EPO-SA-PEG 1 kDa exhibited almost all of the bioactivityof tri-antennary EPO-SA, and di-antennary EPO-SA-PEG 10 kDa exhibitedalmost all of the bioactivity of di-antennary EPO-SA over a range of EPOconcentrations (FIG. 135). Remodeled and glycoPEGylated EPO generated ininsect cells exhibited up to 94% of the in vitro bioactivity of Epogen™,which is EPO expressed in CHO cells without further glycan remodeling orPEGylation (Table 22).

TABLE 22 In vitro activity of the EPO constructs as compared withEpogen ™ at 2 μg/ml protein and 48 hr. Compound (2 μg/ml protein)Activity (percent of Epogen ™) Biantennary-SA 146 Biantennary-SA-PEG 1K94 Biantennary-SA-PEG 10K 75 Triantennary-SA 2,3¹ 42 Triantennary-SA-PEG1K 48 Triantennary-SA-PEG 10K 34 ¹The triantennary-SA 2,3 construct hasthe SA molecule bonded in a 2,3 linkage.

The in vivo pharmacokinetics of glycoPEGylated and non-glycoPEGylatedEPO was determined. GlycoPEGylated and non-glycoPEGylated [I¹²⁵]-labeledEPO was bolus injected into rats and the pharmacokinetics of themolecules were determined. As compared with bi-antennary EPO, the AUC ofbi-antennary EPO-PEG 1 kDa was 1.8 times greater, and the AUC ofbi-antennary EPO-PEG 10 kDa was 11 times greater (FIG. 136). As comparedwith bi-antennary EPO, the AUC of bi-antennary EPO-PEG 1 kDa was 1.6times greater, and the AUC of bi-antennary EPO-PEG 10 kDa was 46 timesgreater (FIG. 136). Therefore, the pharmacokinetics of EPO was greatlyimproved by glycoPEGylation.

The in vivo bioactivity of glycoPEGylated and non-glycoPEGylated EPO wasalso determined by measuring the degree to which the EPO construct couldstimulate reticulocytosis. Reticulocytosis is a measure of the rate ofthe maturation of red blood cell precursor cells into mature red bloodcells (erythrocyte). Eight mice per treatment group were given a singlesubcutaneous injection of 10 μg protein/Kg, and the percentreticulocytes was measured at 96 hours (FIG. 137). Tri- and bi-antennaryPEGylated EPO exhibited greater in vivo bioactivity than non-PEGylatedEPO forms, including Epogen™.

Further determination of in vivo bioactivity of the EPO constructs wasassessed by measuring the hematocrit (the percent of whole blood that iscomprised of red blood cells) of CD-1 female mice 15 days afterintraperitoneal injection three times per week with 2.5 μg peptide/kgbody weight of the EPO construct. The hematocrit increment increasedwith the size of the EPO form, with the 82.7 kDa mono-antennary EPO-PEG20 kDa having a slightly greater activity than the 35.6 kDa NESP(Aranesp™) and about two times the bioactivity of 28.5 kDa Epogen™ (FIG.138).

This example illustrates that the generation of a longer-actingglycoPEGylated EPO is feasible. The pharmacokinetic profile ofglycoPEGylated EPO can be customized by altering the number ofglycoPEGylation sites and the size of the PEG molecule added to alterthe half-life of the peptide in the bloodstream. Finally, glycoPEGylatedEPO retains both in vitro and in vivo bioactivity.

18. Preparation and Bioactivity of Sialylated and PEGylated Mono-, Bi-and Tri-Antennary EPO

This example illustrates the production of glycoPEGylated EPO, inparticular PEGylated EPO having mono-antennary and bi-antennary glycanswith PEG linked thereto. The following EPO variants were produced:mono-antennary PEG (1 kDa), PEG (10 kDa) and PEG (20 kDa); bi-antennary2,3-sialic acid (SA), bi-antennary SA-PEG (1 kDa), bi-antennary SA-PEG(10 kDa); tri-antennary 2,3-SA and tri-antennary 2,6-SA capped with2,3-SA.

Recombinant erythropoietin (rEPO) expressed in insect cells was obtainedfrom Protein Sciences (Lot # 060302, Meridan Conn.). The glycancomposition of this batch of EPO had approximately 98% trimannosyl corestructure. FIG. 139A depicts the HPLC analysis of the released glycansfrom this EPO, with peak “P2” representing the trimannosyl core glycan.FIG. 139B shows the MALDI analysis of the released glycans with thestructures of the released glycans beside the peak they represent.

Mono-Antennary Branching

Several steps were performed to produce the mono-antennary branchedstructure. In brief, the first step was a GnT-I/GalT-1 reaction followedby purification using Superdex-75 chromatography. This reaction adds aGlcNAc moiety to one branch of the tri-mannosyl core, and a galactosemoiety onto the GlcNAc moiety. Branching was extended with the ST3Gal3reaction to add the SA-PEG (10 kDa) moiety or the SA-PEG (20 kDa) moietyonto the terminal galactose moiety. The final purification wasaccomplished using Superdex-200 chromatography (Amersham Biosciences,Arlington Heights, Ill.).

GnT-I/GalT-1 Reaction. The GnT-I and GalT-1 reactions were combined andincubated at 32° C. for 36 hours. The reaction contained 1 mg/mL EPO,100 mM Tris-Cl pH 7.2, 150 mM NaCl, 5 mM MnCl₂, 0.02% NaN₃, 3 mMUDP-GlcNAc, 50 mU/mg GnT-I, 3 mM UDP-Gal, and 200 mU/mg GalT-1. FIG. 140depicts the MALDI analysis of glycans released from EPO after theGnT-I/GalT-1 reaction. Glycan analysis showed approximately 90% of theglycans had the desired mono-antennary branched structure with aterminal galactose moiety.

Superdex 75 Purification. After the GnT-I/GalT1 reaction, EPO waspurified from the enzyme protein contaminants and nucleotide sugarsusing a 1.6 cm×60 cm Superdex-75 gel filtration chromatography (AmershamBiosciences, Arlington Heights, Ill.) in PBS containing 0.02% Tween 20(Sigma-Aldrich Corp., St. Louis, Mo.).

ST3Gal3 Reaction. The ST3Gal3 PEGylation reaction was incubated at 32°C. for 24 hours. The reaction contained 1 mg/mL EPO, 100 mM Tris-Cl pH7.2, 150 mM NaCl, 0.02% NaN₃, 200 mU/mg ST3Gal3, and 0.5 mM CMP-SA-PEG(10 kDa) or 0.5 mM CMP-SA-PEG (20 kDa). FIG. 141 depicts the SDS-PAGEanalysis of EPO after this reaction. The corresponding molecular weightsof the protein bands indicate that the EPO glycans formed by theGnT-I/GalT-1 reaction were completely sialylated with the PEGderivative.

Superdex 200 Purification. EPO then was purified from the contaminantsof the ST3Gal3 reaction by a 1.6 cm×60 cm Superdex-200 gel filtrationchromatography (Amersham Biosciences, Arlington Heights, Ill.) in PBScontaining 0.02% Tween-20.

TF-1 Cell In Vitro Bioassay of Mono-antennary PEGylated EPO. The TF-1cell line is used to assess the activity of EPO in vitro. The TF-1 cellsline is a myeloid progenitor cell line available from the American TypeCulture Collection (Catalogue No. CRL-2003, Rockville, Md.). The cellline is completely dependant on Interleukin-3 or Granulocyte-MacrophageColony-Stimulating Factor for viability. TF-1 cells provide a goodsystem for investigating the effect of EPO on proliferation anddifferentiation.

The TF-1 cells were grown in RPMI with 10% FBS and 12 ng/ml GM-CSF at37° C. in 5% CO₂. The cells were suspended at a concentration of 10,000cells/ml of media. 200 μI aliquots of cells were dispensed into a96-well plate. The cells were incubated with 0.1 to 10 μg/ml EPO for 48hrs.

The MTT viability assay was then performed by first adding 25 μl of 5μg/ml MTT (3-[4,5-dimethlythiazol-2-yl]-2,5-diphenyltetrazolium bromide,or thiazolyl blue; Sigma Chemical Co., St. Louis, Mo., Catalogue No.M5655). The plate was incubated for 4 hrs at 37° C. 100 μl ofisopropanol/HCl solution (100 ml isopropanol and 333 μl HCl 6N) wasadded. The absorbency of the plates was read at 570 nm and either 630 or690 nm, and the reading at either 630 nm or 690 nm was subtracted forthe reading at 570 nm.

FIG. 142 depicts the results of the bioassay of EPO activity afterPEGylation of it mono-antennary glycans. In this bioassay, themono-antennary PEGylated EPO is much less active that a non-PEGylatedEPO (Epogen).

In Vivo Bioassay of Mono-antennary PEGylated EPO—Examination ofHematological Parameters.

The effect of EPO-SA-PEG (10 kDa) and EPO-SA-PEG (20 kDa), thepreparation of which compounds is described in detail above, onhematological parameters of male Sprague Dawley rats was examined.Sprague Dawley rats are a well-studied experimental model and techniquesfor the use and care of such animals are well-known to those skilled inthe art. Therefore, Sprague Dawley rats provide a good system forstudying the clinical and physiological effects of EPO-SA-PEG (10 kDa)and EPO-SA-PEG (20 kDa) according to the present invention.

Preparation, Care, and Handling of Test Subjects. In vivo testing inaccordance with the methods and compositions of the present inventionwas conducted at TherImmune Research Corporation (Gaithersburg, Md.).Test subjects for the in vivo studies included 170 male Sprague Dawleyrats, obtained from Harlan (Harlan Sprague Dawley, Inc., Indianapolis,Ind.). Subjects were all at least 300 grams in weight and at least 70days old at the time of shipment. Subjects were cared for according toTherImmune's Institutional Animal Care and Use Committee, in accordancewith provisions of the USDA Animal Welfare Act, the PHS Policy on HumaneCare and Use of Laboratory Animals, and the U.S. Interagency ResearchAnimal Committee Principles for the Utilization and Care of ResearchAnimals. The study was conducted in accordance with the FDA GoodLaboratory Practice Standards, 21 CFR Part 58. Subjects were housedindividually in polycarbonate cages, under controlled environmentalconditions, and were provided fresh food weekly, ad libitum, and tapwater was made available to test subjects ad libitum via either anautomatic watering system or water bottles.

Subjects were randomly assigned to groups for testing, and the minimumnumber of animals needed to give biologically meaningful data were used.Subjects were dosed via subcutaneous injection, and dosing frequency andquantity is indicated in Table 25, and were observed prior to doseadministration, and at least twice daily for signs of toxicity,moribundity and mortality. Body weights were measured and physicalexaminations were conducted on subjects pre- and post-study, as well ason a weekly basis during the study.

For hematology studies, a 0.25 ml (±15%) sample of blood was collectedfrom the jugular vein of all surviving subjects prior to the first doseon study day one, as well as on study days 3, 5, 8, 10, 12, 15, 17, 19,22, 24, 26 and 29. Hematological analysis of the blood of the testsubjects included examination of cellular morphology, erythrocyte count,hematocrit, hemoglobin, leukocyte, leukocyte differential, mean cellhemoglobin, mean cell hemoglobin concentration, mean cell volume, meanplatelet volume, platelet count, and reticulocytes.

TABLE 25 Summary of test subject grouping and dosage. Dose Dose VolumeConcentration Dose Number of Group Treatment (ug/kg) (ml/kg) (ug/mlFrequency Animals 1 Vehicle — 0.33 — Every 3 days¹ 10 2 P113 2.5 0.337.5 Every 3 days¹ 5 3 P113 6.0 0.33 18 Every 3 days¹ 5 4 P140 2.5 0.337.5 Every 3 days¹ 5 5 P140 6.0 0.33 18 Every 3 days¹ 5 6 P138 2.5 0.337.5 Every 3 days¹ 5 7 P138 6.0 0.33 18 Every 3 days¹ 5 8 P139 2.5 0.337.5 Every 3 days¹ 5 9 P139 6.0 0.33 18 Every 3 days¹ 5 10 Vehicle — 0.33— Every 5 days² 10 11 P140 4 0.33 12 Every 5 days² 5 12 P140 12 0.33 36Every 5 days² 5 13 P138 4 0.33 12 Every 5 days² 5 14 P138 12 0.33 36Every 5 days² 5 15 P139 4 0.33 12 Every 5 days² 5 16 P139 12 0.33 36Every 5 days² 5 17 Vehicle — 0.33 — Every 7 days³ 10 18 P140 6 0.33 18Every 7 days³ 5 19 P140 25 0.33 75 Every 7 days³ 5 20 P138 6 0.33 18Every 7 days³ 5 21 P138 25 0.33 75 Every 7 days³ 5 22 P139 6 0.33 18Every 7 days³ 5 23 P139 25 0.33 75 Every 7 days³ 5 24 Vehicle — 0.33 —Every 10 days⁴ 10 25 P140 7.5 0.33 23 Every 10 days⁴ 5 26 P140 50 0.33150 Every 10 days⁴ 5 27 P138 7.5 0.33 23 Every 10 days⁴ 5 28 P138 500.33 150 Every 10 days⁴ 5 29 P139 7.5 0.33 23 Every 10 days⁴ 5 30 P13950 0.33 150 Every 10 days⁴ 5 Study days are indicated for each group ofsubjects as follows: ¹= SD 1, 4, 7, 10, 13, 16, 19, 22, 25 and 28 ²= SD1, 6, 11, 16, 21 and 27 ³= SD 1, 8, 15 and 22 ⁴= SD 1, 11 and 21

On study day 29, after clinical pathology blood collection, allsurviving subjects were euthanized via carbon dioxide inhalation. Bloodwas collected from the abdominal aorta. Moribund subjects that werepreviously euthanized, as well as subjects found dead during the study,were subjected to full necropsy.

Analysis of results and interpretation of data. Quantitative resultswere analyzed using techniques known to those skilled in the relevantart. In particular, results were analyzed using the Kolmogorov-Smirnovtest for normality, the Levene Median test for equal variance, andone-way Analysis of Variance (ANOVA). In instances where the normalityor equal variance test failed, the analysis was continued using thenon-parametric Kruskal-Wallis ANOVA on rank-transformed data. Parametricdata was analyzed using the Dunnett's t-test to delinieate which groupsdiffered from the control. For non-parametric data, the Dunns test wasused to delineate which groups differed from the control. A probabilityvalue of less than 0.05 (two-tailed) was used as the critical level ofsignificance for all tests.

Based on one analysis of the data from the first fifteen days of thestudy, it was shown that a single dose of 50 μg/kg of glycoPEGylatedmonoantennary EPO, either EPO-SA-PEG (10 kDa) or EPO-SA-PEG (20 kDa)according to the present invention, gives an extended rise in bloodhemoglobin in rats, comparable to the effect of NESP given at the samedose (FIG. 192). Not wishing to be bound by any particular theory, FIG.192 illustrates that EPO-SA-PEG (10 kDa) and EPO-SA-PEG (20 kDa) haveunexpectedly enhanced biological properties.

Bi-Antennary Branching

Several reactions were performed to accomplish the bi-antennarybranching of EPO. Briefly, the first reaction combined the GnT-I andGnT-II reactions to add GlcNAc moieties to two of the tri-mannosyl corebranches. The second reaction, the GalT-1 reaction, adds a galactosemoiety to each GlcNAc moieties. Superdex 75 chromatography (AmershamBiosciences, Arlington Heights, Ill.) was performed prior to the ST3Gal3reaction. The bi-antennary branching was further extended with theST3Gal3 reaction to add either a 2,3-SA, or SA-PEG (1 kDa), SA-PEG (10kDa). Final purification was accomplished using Superdex 200chromatography (Amersham Biosciences, Arlington Heights, Ill.).

GnT-I/GnT-II Reaction. The GNT-I and GNT-II reactions were combined andincubated at 32° C. for 48 hours. The reaction contained 1 mg/mL EPO,100 mM MES pH 6.5, 150 mM NaCl, 20 mM MnCl₂, 0.02% NaN₃, 5 mMUDP-GlcNAc, 100 mU/mg GNT-I, 60 mU/mg GNT-II. The reaction achieved 92%completion of the addition of bi-antennary GlcNAc moieties, with 8%mono-antennary GlcNAc moieties. FIG. 143A shows the HPLC analysis of thereleased glycans, where peak “P3” represents the bi-antennary GlcNAcglycan. FIG. 143B depicts the MALDI analysis of the released glycanswith the structures of the glycans indicated beside the peak that theyrepresent.

In order to further the reaction, an additional 20 mU/mg of GNT-II wasadded along with 1 mM UDP-GlcNAc, 5 mM MnCl₂, 0.02% NaN₃, and the mixurewas incubated for 4 hours at 32° C. Greater than 99% of this reactionachieved completion of the bi-antennary GlcNAc glycan.

GalT-1 Reaction. The GalT-1 reaction was started immediately after thecompletion of the second GnT-II reaction. Enzyme and nucleotide sugarwere added to the completed GnT-II reaction at concentrations of 0.5U/mg GalT-1 and 3 mM UDP-Gal.

When the GalT-1 reaction was performed on a small scale, with about 100μg EPO per reaction, approximately 95% of the reaction produced EPO withbi-antennary terminal galactose moiety. FIG. 144A depicts the HPLCanalysis of the released glycans where peak “P2” is the bi-antennaryglycan with terminal galactose moieties (85% of the glycans), and peak“P1” is the bi-antennary glycan without the terminal galactose moieties(15% of the glycans).

The GalT-1 reaction was also performed on a large scale with about 16 mgof EPO per reaction. FIG. 144B depicts the HPLC analysis of the releaseglycans from the large scale GalT-1 reaction, where peak “P2” is thebi-antennary glycan with terminal galactose moieties, and peak “P1” isthe bi-antennary glycan without the terminal galactose moieties.

Superdex 75 Purification. EPO was then purified from the enzyme proteincontaminants and nucleotide sugars using a 1.6 cm×60 cm Superdex-75 gelfiltration chromatography (Amersham Biosciences, Arlington Heights,Ill.) in PBS containing 0.02% Tween 20 after the GnT-1/GalT1 reaction.FIG. 145 depicts the chromatogram of the Superdex 75 gel filtration,where peak 2 is EPO with bi-antennary glycans with terminal galactosemoieties. FIG. 146 shows SDS-PAGE analysis of the products of eachremodeling step indicating the increase in the molecular weight of EPOwith each remodeling step.

ST3Gal3 Reaction. The ST3Gal3 reaction was incubated at 32° C. for 24hours. The reaction contained 0.5 mg/mL EPO, 100 mM Tris-Cl pH 7.2, 150mM NaCl, 0.02% NaN₃, 100 mU/mg ST3Gal3, and 0.5 mM CMP-SA, 0.5 mMCMP-SA-PEG (1 kDa), or 0.5 mM CMP-SA-PEG (10 kDa). FIG. 147 shows theresults of SDS-PAGE analysis of EPO before and after the ST3Gal3reaction. Based on this SDS-PAGE analysis, bi-antennary EPO containingterminal Gal can no longer be visually detected after each ST3Gal3reaction. All sialylated EPO variants show an increase in size comparedto non-sialylated EPO at the start of the reaction.

Superdex 200 Purification. EPO was purified from the contaminants of theST3Gal3 reactions by a 1.6 cm×60 cm Superdex-200 gel filtrationchromatography (Amersham Biosciences, Arlington Heights, Ill.) in PBScontaining 0.02% Tween-20. Table 23 summaries the distribution of glycanstructures at each remodeling step.

TABLE 23 Summary of glycan structures on EPO after each remodeling step.Starting After GnT-I After 2nd After Glycan Material and GnT-II GnT-IIGalT-1 After ST

0.5%

98.0%

1.0% 8.0% 0.5% 0.5% 0.5%

0.5% 92.0% 99.5% 15.5% 15.5%

84.0% 2.0%

82.0% Diamonds represent fucose, and squares represent GlcNAc, circlesrepresent mannose, open circles represent galactose.Tri-Antennary Branching

Several reactions were performed to accomplish the tri-antennarybranching of EPO. Briefly, the first reaction combined the GnT-I andGnT-II reactions to add a GlcNAc moiety to the two outer tri-mannosylcore branches of the glycan. The second reaction, GnT-V reaction, adds asecond GlcNAc moiety to one of the two outer trimannosyl core branchesso that there are now three GlcNAc moieties. The third reaction, GalT-1reaction, adds a galactose moiety to each terminal GlcNAc moiety. TheEPO products were then separated by Superdex 75 chromatography. Thetri-antennary branching was further extended with the ST3Gal3 reactionto add either a 2,3-SA moiety or a 2,6-SA moiety, and capped with a2,3-SA moiety. Final purification was accomplished using Superdex 75chromatography.

GnT-I/GnT-II Reaction. The GnT-I and GnT-II reactions were combined andincubated at 32° C. for 24 hours. The reaction contained 1 mg/mL EPO,100 mM MES pH 6.5, 150 mM NaCl, 20 mM MnCl₂, 0.02% NaN₃, 5 mM UDPGlcNAc, 50 mU/mg GnT-I and 41 mU/mg GNT-II. The reaction achieved 97%completion of the addition of the bi-antennary GlcNAc moiety, with 3%tri-mannosyl core remaining. FIG. 148 depicts the HPLC analysis of theglycans released from EPO after the GnT-I/GnT-II reaction.

GnT-V Reaction. The GnT-V reaction containing 100 mM MES pH 6.5, 5 mMUDP-GlcNAc, 5 mM MnCl₂, 0.02% NaN₃, 10 mU/mg GnT-V and 1 mg/mL EPO, wasincubated at 32° C. for 24 hours. This reaction adds a GlcNAc moiety toan outer mannose moiety already containing a GlcNAc moiety. FIG. 149depicts the HPLC analysis of the glycans released from EPO after theGnT-V reaction. Approximately 92% the glycans released from EPO were thedesired product, tri-antennary branched EPO with terminal GlcNAcmoieties, based on glycan and MALDI analysis. The remaining 8% of theglycans were bi-antennary branched structures containing terminal GlcNAcmoieties.

GalT-1 Reaction. The GalT-1 reaction containing 100 mM Tris pH 7.2, 150mM NaCl, 5 mM UDP Gal, 100 mU/mg GalT-1, 5 mM MnCl₂, 0.02% NaN₃ and 1mg/mL EPO was incubated at 32° C. for 24 hours. FIG. 150 depicts theHPLC analysis of the glycans released from EPO after this reaction.Glycan and MALDI analysis indicates that 97% of the released glycans hadterminal galactose moieties on the tri-antennary branched structures.The remaining 3% was a bi-antennary structure containing a terminalgalactose.

Superdex 75 Purification. After the GnT-I/GalT1 reaction, EPO waspurified from the enzyme protein contaminants and nucleotide sugarsusing a 1.6 cm×60 cm Superdex-75 gel filtration chromatography (AmershamBiosciences, Arlington Heights, Ill.) in PBS containing 0.02% Tween 20.The purified material was divided into two batches to produce thetri-antennary glycan with terminal 2,6-SA moieties and the tri-antennaryglycan with terminal 2,6-SA moieties capped with 2,6-SA moieties.

ST3Gal3 Reaction. The ST3Gal3 reaction was incubated at 32° C. for 24hours. The reaction contained 1 mg/mL galactosylated EPO, 100 mM Tris-ClpH 7.2, 150 mM NaCl, 0.02% NaN₃, 50 mU/mg ST3Gal3, and 3 mM CMP-SA. FIG.151 depicts the HPLC analysis of glycans released from EPO after thisstep. Based on glycan and MALDI analysis, approximately 80% of thereleased glycans were tri-antennary branched structures with terminal2,3-SA moieties. The remaining 20% of the released glycans werebi-antennary structures with terminal 2,3-SA moieties.

ST6Gal1 sialylation Reaction following the ST3Gal3 Reaction. The ST6Gal1reaction was incubated at 32° C. for 24 hours. The reaction contained 1mg/mL sialylated galactosylated EPO, 100 mM Tris-Cl pH 7.2, 150 mM NaCl,0.02% NaN₃, 50 mU/mg ST6Gal1, and 3 mM CMP-SA. FIG. 152 depicts theresults of HPLC analysis of the glycans released from EPO after theST6Gal1 reaction. Based on glycan and MALDI analysis, approximately 80%of the tri-antennary branched glycans contained terminal 2,3-SAmoieties. The remaining 20% of the glycans were bi-antennary withterminal 2,3-SA moieties.

Superdex 75 Purification. EPO was purified from the contaminants of theST3Gal3 reactions by a 1.6 cm×60 cm Superdex-75 gel filtrationchromatography (Amersham Biosciences, Arlington Heights, Ill.) in PBScontaining 0.02% Tween-20.

Bioassay of Tri-antennary and Bi-antennary Sialylated or PEGylated EPO.The activity of the tri-antennary and bi-antennary sialylated EPOglycoforms, and the PEG 10 kDa and 1 kDa bi-antennary glycoforms wereassayed using the TF-1 cell line and the MTT viability test, asdescribed above. FIG. 153 depicts the results of the MTT cellproliferation assay. At 2 μg/ml EDP, the bi-antennary sialylated EPO hadnearly the activity of the control Epogen, while the tri-antennarysialylated EPO had significanly less activity.

Factor IX

19. GlycoPEGylation of Factor IX Produced in CHO Cells

This example sets forth the preparation of asialoFactor IX and itssialylation with CMP-sialic acid-PEG.

Desialylation of rFactor IX. A recombinant form of Coagulation Factor IX(rFactor IX ) was made in CHO cells. 6000 IU of rFactor IX weredissolved in a total of 12 mL USP H₂O. This solution was transferred toa Centricon Plus 20, PL-10 centrifugal filter with another 6 mL USP H₂O.The solution was concentrated to 2 mL and then diluted with 15 mL 50 mMTris-HCl pH 7.4, 0.15 M NaCl, 5 mM CaCl₂, 0.05% NaN₃ and thenreconcentrated. The dilution/concentration was repeated 4 times toeffectively change the buffer to a final volume of 3.0 mL. Of thissolution, 2.9 mL (about 29 mg of rFactor IX) was transferred to a smallplastic tube and to it was added 530 mU α2-3,6,8-Neuraminidase—agaroseconjugate (Vibrio cholerae, Calbiochem, 450 μL). The reaction mixturewas rotated gently for 26.5 hours at 32° C. The mixture was centrifuged2 minutes at 10,000 rpm and the supernatant was collected. The agarosebeads (containing neuraminidase) were washed 6 times with 0.5 mL 50 mMTris-HCl pH 7.12, 1 M NaCl, 0.05% NaN₃. The pooled washings andsupernatants were centrifuged again for 2 minutes at 10,000 rpm toremove any residual agarose resin. The pooled, desialylated proteinsolution was diluted to 19 mL with the same buffer and concentrated downto ˜2 mL in a Centricon Plus 20 PL-10 centrifugal filter. The solutionwas twice diluted with 15 mL of 50 mM Tris-HCl pH 7.4, 0.15 M NaCl,0.05% NaN₃ and reconcentrated to 2 mL. The final desialyated rFactor IXsolution was diluted to 3 mL final volume (˜10 mg/mL) with the TrisBuffer. Native and desialylated rFactor IX samples were analyzed byIEF-Electrophoresis. Isoelectric Focusing Gels (pH 3-7) were run using1.5 μL (15 μg) samples first diluted with 10 μL Tris buffer and mixedwith 12 μL sample loading buffer. Gels were loaded, run and fixed usingstandard procedures. Gels were stained with Colloidal Blue Stain (FIG.154), showing a band for desialylated Factor IX.

Preparation of PEG (1 kDa and 10 kDa)-SA-Factor IX. DesialylatedrFactor-IX (29 mg, 3 mL) was divided into two 1.5 mL (14.5 mg) samplesin two 15 mL centrifuge tubes. Each solution was diluted with 12.67 mL50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 0.05% NaN₃ and either CMP-SA-PEG-1 kor 10 k (7.25 μmol) was added. The tubes were inverted gently to mix and2.9 U ST3Gal3 (326 μL) was added (total volume 14.5 mL). The tubes wereinverted again and rotated gently for 65 hours at 32° C. The reactionswere stopped by freezing at −20° C. 10 μg samples of the reactions wereanalyzed by SDS-PAGE. The PEGylated proteins were purified on a TosoHaas Biosep G3000SW (21.5×30 cm, 13 um) HPLC column with Dulbecco'sPhosphate Buffered Saline, pH 7.1 (Gibco), 6 mL/min. The reaction andpurification were monitored using SDS Page and IEF gels. NovexTris-Glycine 4-20% 1 mm gels were loaded with 10 μL (10 μg) of samplesafter dilution with 2 μL of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05%NaN₃ buffer and mixing with 12 μL sample loading buffer and 1 μL 0.5 MDTT and heated for 6 minutes at 85° C. Gels were stained with ColloidalBlue Stain (FIG. 155) showing a band for PEG (1 kDa and 10kDa)-SA-Factor IX.

20. Direct Sialyl-GlycoPEGylation of Factor IX

This example sets forth the preparation of sialyl-PEGylation of FactorIX without prior sialidase treatment.

Sialyl-PEGylation of Factor-IX with CMP-SA-PEG-(10 KDa). Factor IX (1100IU), which was expressed in CHO cells and was fully sialylated, wasdissolved in 5 mL of 20 mM histidine, 520 mM glycine, 2% sucrose, 0.05%NaN₃ and 0.01% polysorbate 80, pH 5.0. The CMP-SA-PEG-(10 kDa) (27 mg,2.5 μmol) was then dissolved in the solution and 1 U of ST3Gal3 wasadded. The reaction was complete after gently mixing for 28 hours at 32°C. The reaction was analyzed by SDS-PAGE as described by Invitrogen. Theproduct protein was purified on an Amersham Superdex 200 (10×300 mm, 13μm) HPLC column with phosphate buffered saline, pH 7.0 (PBS), 1 mL/min.R_(t)=9.5 min.

Sialyl-PEGylation of Factor-IX with CMP-SA-PEG-(20 kDa). Factor IX (1100IU), which was expressed in CHO cells and was fully sialylated, wasdissolved in 5 mL of 20 mM histidine, 520 mM glycine, 2% sucrose, 0.05%NaN₃ and 0.01% polysorbate 80, pH 5.0. The CMP-SA-PEG-(20 kDa) (50 mg,2.3 μmol) was then dissolved in the solution and CST-II was added. Thereaction mixture was complete after gently mixing for 42 hours at 32° C.The reaction was analyzed by SDS-PAGE as described by Invitrogen.

The product protein was purified on an Amersham Superdex 200 (10×300 mm,13 μm) HPLC column with phosphate buffered saline, pH 7.0 (Fisher), 1mL/min. R_(t)=8.6 min.

21. Sialic Acid Capping of GlycoPEGylated Factor IX

This examples sets forth the procedure for sialic acid capping ofsialyl-glycoPEGylated peptides. Here, Factor-IX is the exemplarypeptide.

Sialic acid capping of N-linked and O-linked Glycans of Factor-IX-SA-PEG(10 kDa). Purified r-Factor-IX-PEG (10 kDa) (2.4 mg) was concentrated ina Centricon® Plus 20 PL-10 (Millipore Corp., Bedford, Mass.) centrifugalfilter and the buffer was changed to 50 mM Tris-HCl pH 7.2, 0.15 M NaCl,0.05% NaN₃ to a final volume of 1.85 mL. The protein solution wasdiluted with 372 μL of the same Tris buffer and 7.4 mg CMP-SA (12 μmol)was added as a solid. The solution was inverted gently to mix and 0.1 UST3Gal1 and 0.1 U ST3Gal3 were added. The reaction mixture was rotatedgently for 42 hours at 32° C.

A 10 μg sample of the reaction was analyzed by SDS-PAGE. NovexTris-Glycine 4-12% 1 mm gels were performed and stained using ColloidalBlue as described by Invitrogen. Briefly, samples, 10 μL (10 μg), weremixed with 12 μL sample loading buffer and 1 μL 0.5 M DTT and heated for6 minutes at 85° C. (FIG. 156, lane 4).

Factor VIIa

22. GlycoPEGylation of Recombinant Factor VIIa Produced in BHK Cells

This example sets forth the PEGylation of recombinant Factor VIIa madein BHK cells.

Preparation of Asialo-Factor VIIa. Recombinant Factor VIIa was producedin BHK cells (baby hamster kidney cells). Factor VIIa (14.2 mg) wasdissolved at 1 mg/ml in buffer solution (pH 7.4, 0.05 M Tris, 0.15 MNaCl, 0.001 M CaCl₂, 0.05% NaN₃) and was incubated with 300 mU/mLsialidase (Vibrio cholera)-agarose conjugate for 3 days at 32° C. Tomonitor the reaction a small aliquot of the reaction was diluted withthe appropriate buffer and an IEF gel performed according to Invitrogenprocedures (FIG. 157). The mixture was centrifuged at 3,500 rpm and thesupernatant was collected. The resin was washed three times (3×2 mL)with the above buffer solution (pH 7.4, 0.05 M Tris, 0.15 M NaCl, 0.05%NaN₃) and the combined washes were concentrated in a Centricon-Plus-20.The remaining solution was buffer exchanged with 0.05 M Tris (H 7.4),0.15 M NaCl, 0.05% NaN₃ to a final volume of 14.4 mL.

Preparation of Factor VIIa-SA-PEG (1 kDa and 10 kDa). The desialylationrFactor VIIa solution was split into two equal 7.2 ml samples. To eachsample was added either CMP-SA-5-PEG(1 kDa) (7.4 mg) or CMP-SA-5-PEG(10kDa) (7.4 mg). ST3Gal3 (1.58 U) was added to both tubes and the reactionmixtures were incubated at 32° C. for 96 hrs. The reaction was monitoredby SDS-PAGE gel using reagents and conditions described by Invitrogen.When the reaction was complete, the reaction mixture was purified usinga Toso Haas TSK-Gel-3000 preparative column using PBS buffer (pH 7.1)and collecting fractions based on UV absorption. The combined fractionscontaining the product were concentrated at 4° C. in Centricon-Plus-20centrifugal filters (Millipore, Bedford, Mass.) and the concentratedsolution reformulated to yield 1.97 mg (bicinchoninic acid proteinassay, BCA assay, Sigma-Aldrich, St. Louis Mo.) of Factor VIIa-PEG. Theproduct of the reaction was analyzed using SDS-PAGE and IEF analysisaccording to the procedures and reagents supplied by Invitrogen. Sampleswere dialyzed against water and analyzed by MALDI-TOF. FIG. 158 showsthe MALDI results for native Factor VIIa. FIG. 159 contains the MALDIresults for Factor VIIa PEGylated with 1 kDa PEG where peak of FactorVIIa PEGylated with 1 KDa PEG is evident. FIG. 160 contains the MALDIresults for Factor VIIa PEGylated with 10 kDa PEG where a peak forFactor VIIa PEGylated with 10 kDa PEG is evident. FIG. 161 depicts theSDS-PAGE analysis of all of the reaction products, where a band forFactor VIIa-SA-PEG (10 kDa) is evident.

Follicle Stimulating Hormone (FSH)

23. GlycoPEGylation of Human Pituitary-Derived FSH

This example illustrates the assembly of a conjugate of the invention.Follicle Stimulating Hormone (FSH) is desialylated and then conjugatedwith CMP-(sialic acid)-PEG.

Desialylation of Follicle Stimulating Hormone. Follicle StimulatingHormone (FSH) (Human Pituitary, Calbiochem Cat No. 869001), 1 mg, wasdissolved in 500 μL 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 5 mM CaCl₂. Thissolution, 375 μL, was transferred to a small plastic tube and to it wasadded 263 mU Neuraminidase II (Vibrio cholerae). The reaction mixturewas shaken gently for 15 hours at 32° C. The reaction mixture was addedto N-(p-aminophenyl)oxamic acid-agarose conjugate, 600 μL,pre-equilibrated with 50 mM Tris-HCl pH 7.4, 150 mM NaCl and 0.05% NaN₃and gently rotated 6.5 hours at 4° C. The suspension was centrifuged for2 minutes at 14,000 rpm and the supernatant was collected. The beadswere washed 5 times with 0.5 mL of the buffer and all supernatants werepooled. The enzyme solution was dialyzed (7000 MWCO) for 15 hours at 4°C. with 2 L of a solution containing 50 mM Tris-HCl pH 7.4, 1 M NaCl,0.05% NaN₃, and then twice for 4 hours at 4 ° C. into 50 mM Tris-HCl pH7.4, 1 M NaCl, 0.05% NaN₃. The solution was concentrated to 2 μg/μL bySpeed Vac and stored at −20° C. Reaction samples were analyzed by IEFgels (pH 3-7) (Invitrogen) (FIG. 162).

Preparation of human pituitary-derived SA-FSH and PEG-SA-FollicleStimulating Hormone. Desialylated FSH (100 μg, 50 μL) and CMP-sialicacid or CMP-SA-PEG (1 kDa or 10 kDa) (0.05 umol) were dissolved in 13.5μL H₂O (adjusted to pH 8 with NaOH) in 0.5 mL plastic tubes. The tubeswere vortexed briefly and 40 mU ST3Gal3 (36.5 μL) was added (totalvolume 100 μL). The tubes were vortexed again and shaken gently for 24hours at 32° C. The reactions were stopped by freezing at −80° C.Reaction samples of 15 μg were analyzed by SDS-PAGE (FIG. 163), IEF gels(FIG. 164) and MALDI-TOF. Native FSH was also analyzed by SDS-PAGE (FIG.165)

Analysis of SDS PAGE and IEF Gels of Reaction Products. NovexTris-Glycine 8-16% 1 mm gels for SDS PAGE analysis were purchased fromInvitrogen. 7.5 μL (15 μg) of FSH reaction samples were diluted with 5μL of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% NaN₃ buffer, mixed with15 μL sample loading buffer and 1 μL 9 M μ-mercaptoethanol and heatedfor 6 minutes at 85° C. Gels were run as directed by Invitrogen andstained with Colloidal Blue Stain (Invitrogen).

FSH samples (15 μg) were diluted with 5 μL Tris buffer and mixed with 15μL sample loading buffer (FIG. 162). The samples were then applied toIsoelectric Focusing Gels (pH 3-7) (Invitrogen) (FIG. 165). Gels wererun and fixed as directed by Invitrogen and then stained with ColloidalBlue Stain.

24. GlycoPEGylation of Recombinant FSH Produced Recombinantly in CHOCells

This example illustrates the assembly of a conjugate of the invention.Desialylated FSH was conjugated with CMP-(sialic acid)-PEG.

Preparation of recombinant Asialo-Follicle Stimulation Hormone.Recombinant Follicle Stimulation Hormone (rFSH) produced from CHO wasused in these studies. The 7,500 IU of rFSH was dissolved in 8 mL ofwater. The FSH solution was dialyzed in 50 mM Tris-HCl pH 7.4, 0.15 MNaCl, 5 mM CaCl₂ and concentrated to 500 μL in a Centricon Plus 20centrifugal filter. A portion of this solution (400 μL) (˜0.8 mg FSH)was transferred to a small plastic tube and to it was added 275 mUNeuraminidase II (Vibrio cholerae). The reaction mixture was mixed for16 hours at 32° C. The reaction mixture was added to prewashedN-(p-aminophenyl)oxamic acid-agarose conjugate (800 μL) and gentlyrotated for 24 hours at 4° C. The mixture was centrifuged at 10,000 rpmand the supernatant was collected. The beads were washed 3 times with0.6 mL Tris-EDTA buffer, once with 0.4 mL Tris-EDTA buffer and once with0.2 mL of the Tris-EDTA buffer and all supernatants were pooled. Thesupernatant was dialyzed at 4° C. against 2 L of 50 mM Tris-HCl pH 7.4,1 M NaCl, 0.05% NaN₃ and then twice more against 50 mM Tris-HCl pH 7.4,1 M NaCl, 0.05% NaN₃. The dialyzed solution was then concentrated to 420μL in a Centricon Plus 20 centrifugal filter and stored at μ20° C.

Native and desialylated rFSH samples were analyzed by SDS-PAGE and IEF(FIG. 166). Novex Tris-Glycine 8-16% 1 mm gels were purchased fromInvitrogen. Samples (7.5 μL, 15 μg) samples were diluted with 5 μL of 50mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% NaN₃ buffer, mixed with 15 μLsample loading buffer and 1 μL 9 M β-mercaptoethanol and heated for 6minutes at 85° C. Gels were run as directed by Invitrogen and stainedwith Colloidal Blue Stain (Invitrogen). Isoelectric Focusing Gels (pH3-7) were purchased from Invitrogen. Samples (7.5 μL, 15 μg) werediluted with 5 μL Tris buffer and mixed with 15 μL sample loadingbuffer. Gels were loaded, run and fixed as directed by Invitrogen. Gelswere stained with Colloidal Blue Stain. Samples of native anddesialylated FSH were also dialyzed against water and analyzed byMALDI-TOF.

Sialyl-PEGylation of recombinant Follicle Stimulation Hormone.Desialylated FSH (100 μg, 54 μL) and CMP-SA-PEG (1 kDa or 10 kDa) (0.05μmol) were dissolved in 28 μL 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃,pH 7.2 in 0.5 nL plastic tubes. The tubes were vortexed briefly and 20mU of ST3Gal3 was added (total volume 100 μL). The tubes were vortexedagain, mixed gently for 24 hours at 32° C. and the reactions stopped byfreezing at −80° C. Samples of this reaction were analyzed as describedabove by SDS-PAGE gels (FIG. 167), IEF gels (FIG. 168) and MALDI-TOF MS.

MALDI was also performed on the PEGylated rFSH. During ionization,SA-PEG is eliminated from the N-glycan structure of the glycoprotein.Native FSH gave a peak at 13928; AS-rFSH (13282); resialylated r-FSH(13332); PEG1000-rFSH (13515; 14960 (1); 16455 (2); 17796 (3); 19321(4)); and PEG 10000 (23560 (1); 34790 (2); 45670 (30; and 56760 (4)).

25. Pharmacokinetic Study of GlycoPEGylated FSH

This example sets forth the in vivo testing of the pharmacokineticproperties glycoPEGylated Follicle Stimulating Hormone (FSH) preparedaccording to the methods of the invention as compared to non-PEGylatedFSH.

FSH, FSH-SA-PEG (1 kDa) and FSH-SA-PEG (10 kDa) were radioiodinatedusing standard conditions (Amersham Biosciences, Arlington Heights,Ill.) and formulated in phosphate buffered saline containing 0.1% BSA.After dilution in phosphate buffer to the appropriate concentration,each of the test FSH proteins (0.4 μg, each) was injected intraveneouslyinto female Sprague Dawley rats (250-300 g body weight) and blood drawnat time points from 0 to 80 hours. Radioactivity in blood samples wasanalyzed using a gamma counter and the pharmacokinetics analyzed usingstandard methods (FIG. 169). FSH was cleared from the blood much morequickly than FSH-PEG(1 kDa), which in turn was clear somewhat morequickly than FSH-PEG(10 kDa).

26. Sertoli Cell Bioassay for In Vitro Activity of GlycoPEGylated FSH

This example sets forth a bioassay for follicle stimulating hormone(FSH) activity based on cultured Sertoli cells. This assay is useful todetermine the bioactivity of FSH after glycan remodeling, includingglycoconjugation.

This bioassay is based on the dose-response relationship that existsbetween the amount of estradiol produced when FSH, but not lutenizinghormone (LH), is added to cultured Sertoli cells obtained from immatureold rats. Exogenous testosterone is converted to 17β-estradiol in thepresence of FSH.

Seven to 10 days old Sprague-Dawley rats were used to obtain Sertolicells. After sacrifice, testes were decapsulated and tissue wasdispersed by incubation in collagenase (1 mg/ml), trypsin (1 mg/ml),hyaluronidase (1 mg/ml) and DNases (5 μg/ml) for 5 to 10 min. The tubulefragments settled to the bottom of the flask and were washed in PBS(1×). The tubule fragments were reincubated for 20 min with a mediacontaining the same enzymes: collagenase (1 mg/ml), trypsin (1 mg/ml),hyaluronidase (1 mg/ml) and DNases (5 μg/ml).

The tubule fragments were homogenized and plated into a 24 well plate ina serum free media. 5×10⁵ cells were dispersed per well. After 48 hincubation at 37° C. and 5% CO₂, fresh media was added to the cells.Composition of the serum free media: DMEM (1 vol), Ham's F10 nutrientmixture (1 vol), insulin 1 μg/ml, Transferrin 5 μg/ml, EGF 10 ng/ml, T420 pg/ml, Hydrocortisone 10⁻⁸M, Retinoic acid 10⁻⁶M.

The stimulation experiment consists of a 24 hour incubation withstandard FSH or samples at 37° C. and 5% CO₂. The mean intra-assaycoefficient of variation is 9% and the mean inter-assay coefficient ofvariation is 11%.

The 17B-estradiol Elisa Kit DE2000 (R&D Systems, Minneapolis, Minn.) wasused to quantify the level of estradiol after incubation with FSH,FSH-SA-PEG (1 kDa) and FSH-SA-PEG (10 kDa).

The procedure was as follows: 100 μl of Estradiol Standard (providedwith kit and prepared as per instructions with kit) or sample waspipetted into wells of 17B-estradiol Elisa plate(s); 50 μl of17B-estradiol Conjugate (provided with kit, prepared as per instructionswith kit) was added to each well; 50 μl of 17B-estradiol antibodysolution (provided with kit and prepared as per instructions with kit)was added to each well; plates were incubated for 2 hour at roomtemperature at 200 rpm; the liquid was aspirated from each well; thewells were washed 4 times using the washing solution; all the liquid wasremoved from the wells; 200 μl of pNPP Substrate (provided with kit andprepared as per instructions with kit) was added to all wells andincubated for 45 min; 50 μl of Stop solution (provided with kit andprepared as per instructions with kit) was added and the plates wereread it at 405 nm (FIG. 170). While FSH-PEG(10 kDa) exhibited a modeststimulation of Sertoli cells, at 1 μg/ml, FSH-PEG(1 kDa) stimulatedSertoli cells up to 50% more than unPEGylated FSH.

27. Steelman-Pohley Bioassay of In Vivo Activity of GlycoPEGylated FSH

In this example, the Steelman-Pohley bioassay (Steelman and Pohley,1953, Endocrinology 53:604-615) was used to determine the in vivoactivity of glycoPEGylated FSH. The Steelman-Pohley assay uses thechange in ovary weight of a rat to measure the in vivo activity of FSHthat is coinjected with human chorionic gonadotropin.

The Steelman-Pohley bioassay was performed according to the protocoldescribed in Christin-Maitre et al. (2000, Methods 21:51-57). Seventyfemale Sprague-Dawley Rats (Charles River Laboratories, Wilmington,Mass.), aged 21 to 22 days, were housed in the testing facility for atleast 5 days before the beginning the assay procedure. Throughout theprocedure, the animal room was climate controlled at 18 to 26° C., 30 to70% relative humidity, and 12 hr. artificial light/12 hr. dark. Allanimals were fed Certified Rodent Chow (Harlan Teklad, Madison Wis.) orthe equivalent, and water, both ad libitum. Animal procedures wereperformed at Calvert Preclinical Services, Inc. (Olyphant, Pa.).

Recombinant FSH was expressed in CHO cells, purified by standardtechniques and glycoPEGylated with PEG (1 kDa). The rats were dividedinto seven test groups, with ten animals per group. On days −1 and 0,animals of all groups were subcutaneously injected with 20 I.U. of humanchorionic gonadotropin (HCG) in 0.5 ml of 0.9% NaCl. On days 1, 2 and 3,the control animals were subcutaneously injected with a dose of 0.5 mlcontaining 20 I.U. HCG in 0.9% NaCl, while in the other groups, the HCGdose was augmented with either rFSH or rFSH-SA-PEG (1 kDa) at either0.14 μg, 0.4 μg or 1.2 μg per dose. On day 4, the animals wereeuthanized by CO₂ inhalation. The ovaries were removed, trimmed andweighted. The average ovary weight was determined for each group.

FIG. 171 presents the average ovary weight of the test groups on day 4.The groups receiving HCG alone (control) or the low dose (0.14 μg) ofeither rFSH or rFSH-SA-PEG (1 kDa) had ovary weights that were roughlyequivalent. The groups receiving the medium (0.4 μg) or high (1.2 μg)doses of rFSH or rFSH-SA-PEG (1 kDa) had ovary weights roughly twicethat of the control group. At the medium dose (0.4 μg), theglycoPEGylated rFSH had roughly the same in vivo activity (as determinedby ovary weight) as the unPEGylated rFSH. At the high dose (1.2 μg), theglycoPEGylated rFSH had somewhat higher in vivo activity than theunPEGylated rFSH.

G-CSF

28. GlycoPEGylation of G-CSF Produced in CHO Cells

Preparation of Asialo-Granulocyte-Colony Stimulation Factor (G-CSF).G-CSF produced in CHO cells is dissolved at 2.5 mg/mL in 50 mM Tris 50mM Tris-HCl pH 7.4, 0.15 M NaCl, 5 mM CaCl₂ and concentrated to 500 μLin a Centricon Plus 20 centrifugal filter. The solution is incubatedwith 300 mU/mL Neuraminidase II (Vibrio cholerae) for 16 hours at 32° C.To monitor the reaction a small aliquot of the reaction is diluted withthe appropriate buffer and a IEF gel performed. The reaction mixture isthen added to prewashed N-(p-aminophenyl)oxamic acid-agarose conjugate(800 μL/mL reaction volume) and the washed beads gently rotated for 24hours at 4° C. The mixture is centrifuged at 10,000 rpm and thesupernatant was collected. The beads are washed 3 times with Tris-EDTAbuffer, once with 0.4 mL Tris-EDTA buffer and once with 0.2 mL of theTris-EDTA buffer and all supernatants are pooled. The supernatant isdialyzed at 4° C. against 50 mM Tris-HCl pH 7.4, 1 M NaCl, 0.05% NaN₃and then twice more against 50 rnM Tris-HCl pH 7.4, 1 M NaCl, 0.05%NaN₃. The dialyzed solution is then concentrated using a Centricon Plus20 centrifugal filter and stored at −20° C. The conditions for the IEFgel were run according to the procedures and reagents provided byInvitrogen. Samples of native and desialylated G-CSF are dialyzedagainst water and analyzed by MALDI-TOF MS.

Preparation of G-CSF-(alpha2,3)-Sialyl-PEG. Desialylated G-CSF wasdissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH7.2. The solution is incubated with 1 mM CMP-sialic acid-PEG and 0.1U/mL of ST3Gall at 32° C. for 2 days. To monitor the incorporation ofsialic acid-PEG, a small aliquot of the reaction hadCMP-SA-PEG-fluorescent ligand added; the label incorporated into thepeptide is separated from the free label by gel filtration on a TosoHaas G3000SW analytical column using PBS buffer (pH 7.1). Thefluorescent label incorporation into the peptide is quantitated using anin-line fluorescent detector. After 2 days, the reaction mixture ispurified using a Toso Haas G3000SW preparative column using PBS buffer(pH 7.1) and collecting fractions based on UV absorption. The product ofthe reaction is analyzed using SDS-PAGE and IEF analysis according tothe procedures and reagents supplied by Invitrogen. Samples of nativeand PEGylated G-CSF are dialyzed against water and analyzed by MALDI-TOFMS.

Preparation of G-CSF-(alpha2,8)-Sialyl-PEG. G-CSF produced in CHO cells,which contains an alpha2,3-sialylated O-linked glycan, is dissolved at2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH 7.2. Thesolution is incubated with 1 mM CMP-sialic acid-PEG and 0.1 U/mL ofCST-II at 32° C. for 2 days. To monitor the incorporation of sialicacid-PEG, a small aliquot of the reaction has CMP-SA-PEG-fluorescentligand added; the label incorporated into the peptide is separated fromthe free label by gel filtration on a Toso Haas G3000SW analyticalcolumn using PBS buffer (pH 7.1). The fluorescent label incorporationinto the peptide is quantitated using an in-line fluorescent detector.After 2 days, the reaction mixture is purified using a Toso Haas G3000SWpreparative column using PBS buffer (pH 7.1) and collecting fractionsbased on UV absorption. The product of the reaction is analyzed usingSDS-PAGE and IEF analysis according to the procedures and reagentssupplied by Invitrogen. Samples of native and PEGylated G-CSF aredialyzed against water and analyzed by MALDI-TOF MS.

Preparation of G-CSF-(alpha2,6)-Sialyl-PEG. G-CSF, containing onlyO-linked GalNAc, is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 MNaCl, 0.05% NaN₃, pH 7.2. The solution is incubated with 1 mM CMP-sialicacid-PEG and 0.1 U/mL of ST6GalNAcI or II at 32° C. for 2 days. Tomonitor the incorporation of sialic acid-PEG, a small aliquot of thereaction has CMP-SA-PEG-fluorescent ligand added; the label incorporatedinto the peptide is separated from the free label by gel filtration on aToso Haas G3000SW analytical column using PBS buffer (pH 7.1). Thefluorescent label incorporation into the peptide is quantitated using anin-line fluorescent detector. After 2 days, the reaction mixture ispurified using a Toso Haas G3000SW preparative column using PBS buffer(pH 7.1) and collecting fractions based on UV absorption. The product ofthe reaction is analyzed using SDS-PAGE and IEF analysis according tothe procedures and reagents supplied by Invitrogen. Samples of nativeand PEGylated G-CSF are dialyzed against water and analyzed by MALDI-TOFMS.

G-CSF produced in CHO cells was treated with Arthrobacter sialidase andwas then purified by size exclusion on Superdex75 and was treated withST3Gal1 or ST3 Gal2 and then with CMP-SA-PEG 20 Kda. The resultingmolecule was purified by ion exchange and gel filtration and analysis bySDS PAGE demonstrated that the PEGylation was complete. This is thefirst demonstration of glycoPEGylation of an O-linked glycan.

Glucocerebrosidase

29. Glucocerebrosidase-Mannose-6-Phosphate Produced in CHO Cells

This example sets forth the procedure to glycoconjugatemannose-6-phosphate to a peptide produced in CHO cells such asglucocerebrosidase.

Preparation of asialo-glucoceramidase. Glucocerebrosidase produced inCHO cells is dissolved at 2.5 mg/mL in 50 mM Tris 50 mM Tris-HCl pH 7.4,0.15 M NaCl, and is incubated with 300 mU/mL sialidase-agarose conjugatefor 16 hours at 32° C. To monitor the reaction a small aliquot of thereaction is diluted with the appropriate buffer and a IEF gel andSDS-PAGE performed according to Invitrogen procedures. The mixture iscentrifuged at 10,000 rpm and the supernatant is collected. The beadsare washed 3 times with Tris-EDTA buffer, once with 0.4 mL Tris-EDTAbuffer, and once with 0.2 mL of the Tris-EDTA buffer. All supernatantsare pooled. The supernatant is dialyzed at 4° C. against 50 mM Tris-HClpH 7.4, 1 M NaCl, 0.05% NaN₃ and then twice more against 50 mM Tris-HClpH 7.4, 1 M NaCl, 0.05% NaN₃. The dialyzed solution is then concentratedusing a Centricon Plus 20 centrifugal filter. The product of thereaction is analyzed using SDS-PAGE and IEF analysis according to theprocedures and reagents supplied by Invitrogen. Samples are dialyzedagainst water and analyzed by MALDI-TOF MS.

Preparation of Glucocerebrosidase-SA-linker-Mannose-6-phosphate(procedure 1). Asialo-glucocerebrosidasefrom above is dissolved at 2.5mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH 7.2. The solutionis incubated with 1 mM CMP-sialic acid-linker-Man-6-phosphate and 0.1U/mL of ST3Gal3 at 32° C. for 2 days. To monitor the incorporation ofsialic acid-linker-Man-6-phosphate, a small aliquot of the reaction hadCMP-SA-PEG-fluorescent ligand added; the label incorporated into thepeptide is separated from the free label by gel filtration on a TosoHaas TSK-Gel-3000 analytical column using PBS buffer (pH 7.1). Thefluorescent label incorporation into the peptide is quantitated using anin-line fluorescent detector. When the reaction is complete, thereaction mixture is purified using a Toso Haas TSK-Gel-3000 preparativecolumn using PBS buffer (pH 7.1) and collecting fractions based on UVabsorption. The product of the reaction is analyzed using SDS-PAGE andIEF analysis according to the procedures and reagents supplied byInvitrogen. Samples are dialyzed against water and analyzed by MALDI-TOFMS.

Preparation of Glucocerebrosidase-SA-linker-Mannose-6-phosphate(procedure 2). Glucocerebrosidase, produced in CHO but incompletelysialylated, is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl,0.05% NaN₃, pH 7.2. The solution is incubated with 1 mM CMP-sialicacid-linker-Man-6-phosphate and 0.1 U/mL of ST3Gal3 at 32° C. for 2days. To monitor the incorporation of sialicacid-linker-Man-6-phosphate, a small aliquot of the reaction hadCMP-SA-PEG-fluorescent ligand added; the label incorporated into thepeptide is separated from the free label by gel filtration on a TosoHaas TSK-Gel-3000 analytical column using PBS buffer (pH 7.1). Thefluorescent label incorporation into the peptide is quantitated using anin-line fluorescent detector. When the reaction is complete, thereaction mixture is purified using a Toso Haas TSK-Gel-3000 preparativecolumn using PBS buffer (pH 7.1) and collecting fractions based on UVabsorption. The product of the reaction is analyzed using SDS-PAGE andIEF analysis according to the procedures and reagents supplied byInvitrogen. Samples are dialyzed against water and analyzed by MALDI-TOFMS.

30. Glucocerebrosidase-Transferrin

This example sets forth the procedures for the glycoconjugation ofproteins, and in particular, transferrin is glycoconjugated toglucocerebrosidase. The GlcNAc-ASN structures are created onglucoceraminidase, and Transferrin-SA-Linker-Gal-UDP is conjugated toGNDF GlcNAc-ASN structures using galactosyltransferase.

Preparation of GlcNAc-glucocerebrosidase (Cerezyme™). Cerezyme™(glucocerebrosidase) produced in CHO cells is dissolved at 2.5 mg/mL in50 mM Tris 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, and is incubated with 300mU/mL Endo-H-agarose conjugate for 16 hours at 32° C. To monitor thereaction a small aliquot of the reaction is diluted with the appropriatebuffer and a IEF gel and SDS-PAGE performed according to Invitrogenprocedures. The mixture is centrifuged at 10,000 rpm and the supernatantis collected. The beads are washed 3 times with Tris-EDTA buffer, oncewith 0.4 mL Tris-EDTA buffer and once with 0.2 mL of the Tris-EDTAbuffer and all supernatants are pooled. The supernatant is dialyzed at4° C. against 50 mM Tris-HCl pH 7.4, 1 M NaCl, 0.05% NaN₃ and then twicemore against 50 mM Tris-HCl pH 7.4, 1 M NaCl, 0.05% NaN₃. The dialyzedsolution is then concentrated using a Centricon Plus 20 centrifugalfilter. The product of the reaction is analyzed using SDS-PAGE and IEFanalysis according to the procedures and reagents supplied byInvitrogen. Samples are dialyzed against water and analyzed by MALDI-TOFMS.

Preparation of Transferrin-SA-Linker-Gal-glucocerebrosidase.Transferrin-SA-Linker-Gal-UDP from above is dissolved at 2.5 mg/mL in 50mM Tris-HCl, 0.15 M NaCl, 5 mM MnCl₂, 0.05% NaN₃, pH 7.2. The solutionis incubated with 2.5 mg/mL GlcNAc-glucocerebrosidaseand 0.1 U/mL ofgalactosyltransferase at 32° C. for 2 days. To monitor the incorporationof glucocerebrosidase, the peptide is separated by gel filtration on aToso Haas G3000SW analytical column using PBS buffer (pH 7.1) and theproduct detected by UV absorption. The reaction mixture is then purifiedusing a Toso Haas G3000SW preparative column using PBS buffer (pH 7.1)collecting fractions based on UV absorption. The product of the reactionis analyzed using SDS-PAGE and IEF analysis according to the proceduresand reagents supplied by Invitrogen. Samples are dialyzed against waterand analyzed by MALDI-TOF MS.

GM-CSF

31. Generation and PEGylation of GlcNAc-ASN Structures: GM-CSF Producedin Saccharonzyces

This example sets forth the preparation of Tissue-type Activator withPEGylated GlcNAc-Asn structures.

Recombinant GM-CSF expressed in yeast is expected to contain 2 N-linkedand 2 O-linked glycans. The N-linked glycans should be of the branchedmannan type. This recombinant glycoprotein is treated with anendoglycosidase from the group consisting of endoglycosidase H,endoglycosidase-F1, endoglycosidase-F2, endoglycosidase-F3,endoglycosidase-M either alone or in combination with mannosidases I, IIand III to generate GlcNAc nubs on the asparagine (Asn) residues on thepeptide/protein backbone.

The GlcNAc-Asn structures on the peptide/protein backbone is then bemodified with galactose or galactose-PEG using UDP-galactose orUDP-galactose-6-PEG, respectively, and a galactosyltransferase such asGalT1. In one case the galactose-PEG is the terminal residue. In thesecond case the galactose is further modified with SA-PEG using aCMP-SA-PEG donor and a sialyltransferase such as ST3GalIII. In anotherembodiment the GlcNAc-Asn structures on the peptide/protein backbone canbe galactosylated and sialylated as described above, and then furthersialylated using CMP-SA-PEG and an α2,8-sialyltranferase such as theenzyme encoded by the Campylobacter jejuni cst-II gene.

Herceptin™

32. Glycoconjugation of Mithramycin to Herceptin™

This example sets forth the procedures to glycoconjugate a smallmolecule, such as mithramycin to Fc region glycans of an antibodymolecule produced in mammalian cells. Here, the antibody Herceptin™ isused, but one of skill in the art will appreciate that the method can beused with many other antibodies.

Preparation of Herceptin™-Gal-linker-mithramycin. Herceptin™ isdissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 5 mM MnCl₂, 0.05%NaN₃, pH 7.2. The solution is incubated with 1 mMUDP-galactose-linker-mithramycin and 0.1 U/mL of galactosyltransferaseat 32° C. for 2 days to introduce the mithramycin in the Fc regionglycans. To monitor the incorporation of galactose, a small aliquot ofthe reaction has ¹⁴C-galactose-UDP ligand added; the label incorporatedinto the peptide is separated from the free label by gel filtration on aToso Haas G3000SW analytical column using PBS buffer (pH 7.1). Theradioactive label incorporation into the peptide is quantitated using anin-line radiation detector.

When the reaction is complete, the reaction mixture is purified using aToso Haas TSK-Gel-3000 preparative column using PBS buffer (pH 7.1) andcollecting fractions based on UV absorption. The fractions containingproduct are combined, concentrated, buffer exchanged and thenfreeze-dried. The product of the reaction is analyzed using SDS-PAGE andIEF analysis according to the procedures and reagents supplied byInvitrogen. Samples are dialyzed against water and analyzed by MALDI-TOFMS.

Interferon α and Interferon β

33. GlycoPEGylation of Proteins Expressed in Mammalian or InsectSystems: EPO, Interferon α and Interferon β

This example sets forth the preparation of PEGylated peptides that areexpressed in mammalian and insect systems.

Preparation of acceptor from mammalian expression systems. The peptidesto be glycoPEGylated using CMP-sialic acid PEG need to have glycansterminating in galactose. Most peptides from mammalian expressionsystems will have terminal sialic acid that first needs to be removed.

Sialidase digestion. The peptide is desialylated using a sialidase. Atypical procedure involves incubating a 1 mg/mL solution of the peptidein Tris-buffered saline, pH 7.2, with 5 mM CaCl₂ added, with 0.2 U/mLimmobilized sialidase from Vibrio cholera (Calbiochem) at 32° C. for 24hours. Microbial growth can be halted either by sterile filtration orthe inclusion of 0.02% sodium azide. The resin is then removed bycentrifugation or filtration, and then washed to recover entrappedpeptide. At this point, EDTA may be added to the solution to inhibit anysialidase that has leached from the resin.

Preparation from insect expression systems. EPO, interferon-alpha, andinterferon-beta may also be expressed in non-mammalian systems such asyeast, plants, or insect cells. The peptides to be glycoPEGylated usingCMP-sialic acid PEG need to have glycans terminating in galactose. Themajority of the N-glycans on peptides expressed in insect cells, forexample, are the trimannosyl core. These glycans are first built out toglycans terminating in galactose before they are acceptors forsialyltransferase.

Building acceptor glycans from trimannosyl core. Peptide (1 mg/mL) inTris-buffered saline, pH 7.2, containing 5 mM MnCl₂, 5 mM UDP-glcNAc,0.05 U/mL GLCNACT I, 0.05 U/mL GLCNACT II, is incubated at 32° C. for 24hours or until the reaction is substantially complete. Microbial growthcan be halted either by sterile filtration or the inclusion of 0.02%sodium azide. After buffer exchange to remove UDP and other smallmolecules, UDP-galactose and MnCl₂ are each added to 5 mM,galactosyltransferase is added to 0.05 U/mL, and is incubated at 32° C.for 24 H or until the reaction is substantially complete. Microbialgrowth can be halted either by sterile filtration or the inclusion of0.02% sodium azide. The peptides are then ready for glycoPEGylation.

Building O-linked glycans. A similar strategy may be employed forinterferon alpha to produce enzymatically the desired O-glycanGal-GalNAc. If necessary, GalNAc linked to serine or threonine can beadded to the peptide using appropriate peptide GaINAc transferases (e.g.GalNAc T1, GalNAc T2, T3, T4, etc. ) and UDP-GalNAc. Also, if needed,galactose can be added using galactosyltransferase and UDP-galactose.

GlycoPEGylation using sialyltransferase. The glycopeptides (1 mg/mL)bearing terminal galactose in Tris buffered saline +0.02% sodium azideare incubated with CMP-SA-PEG (0.75 mM) and 0.4 U/mL sialyltransferase(ST3Gal3 or ST3Gal4 for N-glycans on EPO and interferon beta; ST3Gal4,or ST3Gal1 for O-glycans on interferon alpha) at 32° C. for 24 hours.Other transferases that may work include the 2,6 sialyltransferase fromPhotobacterium damsella. The acceptor peptide concentration is mostpreferably in the range of 0.1 mg/mL up to the solubility limit of thepeptide. The concentration of CMP-SA-PEG should be sufficient for thereto be excess over the available sites, but not so high as to causepeptide solubility problems due to the PEG, and may range from 50 μM upto 5 mM, and the temperature may range from 2° C. up to 40° C. The timerequired for complete reaction will depend on the temperature, therelative amounts of enzyme to acceptor substrate, the donor substrateconcentration, and the pH.

34. GlycoPEGylation of Interferon a Produced in CHO Cells

Preparation of Asialo-Interferon α. Interferon alpha produced from CHOcells is dissolved at 2.5 mg/mL in 50 mM Tris 50 mM Tris-HCl pH 7.4,0.15 M NaCl, 5 mM CaCl₂ and concentrated to 500 μL in a Centricon Plus20 centrifugal filter. The solution is incubated with 300 mU/mLNeuraminidase II (Vibrio cholerae) for 16 hours at 32° C. To monitor thereaction a small aliquot of the reaction is diluted with the appropriatebuffer and a IEF gel performed. The reaction mixture is then added toprewashed N-(p-aminophenyl)oxamic acid-agarose conjugate (800 μL/mLreaction volume) and the washed beads gently rotated for 24 hours at 4°C. The mixture is centrifuged at 10,000 rpm and the supernatant wascollected. The beads are washed 3 times with Tris-EDTA buffer, once with0.4 mL Tris-EDTA buffer and once with 0.2 mL of the Tris-EDTA buffer andall supernatants were pooled. The supernatant is dialyzed at 4° C.against 50 mM Tris-HCl pH 7.4, 1 M NaCl, 0.05% NaN₃ and then twice moreagainst 50 mM Tris-HCl pH 7.4, 1 M NaCl, 0.05% NaN₃. The dialyzedsolution is then concentrated using a Centricon Plus 20 centrifugalfilter and stored at −20° C. The conditions for the IEF gel are runaccording to the procedures and reagents provided by Invitrogen. Samplesof native and desialylated G-CSF are dialyzed against water and analyzedby MALDI-TOF MS.

Preparation of Interferon-alpha-(alpha2,3)-Sialyl-PEG. Desialylatedinterferon-alpha is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 MNaCl, 0.05% NaN₃, pH 7.2. The solution is incubated with 1 mM CMP-sialicacid-PEG and 0.1 U/mL of ST3Gal1 at 32° C. for 2 days. To monitor theincorporation of sialic acid-PEG, a small aliquot of the reaction hadCMP-SA-PEG-fluorescent ligand added; the label incorporated into thepeptide is separated from the free label by gel filtration on a TosoHaas G3000SW analytical column using PBS buffer (pH 7.1). Thefluorescent label incorporation into the peptide is quantitated using anin-line fluorescent detector. After 2 days, the reaction mixture ispurified using a Toso Haas G3000SW preparative column using PBS buffer(pH 7.1) and collecting fractions based on UV absorption. The product ofthe reaction is analyzed using SDS-PAGE and IEF analysis according tothe procedures and reagents supplied by Invitrogen. Samples of nativeand desialylated Interferon-alpha are dialyzed against water andanalyzed by MALDI-TOF MS.

Preparation of Interferon-alpha-(alpha2,8)-Sialyl-PEG. Interferon-alphaproduced in CHO, which contains an alpha2,3-sialylated O-linked glycan,is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH7.2. The solution is incubated with 1 mM CMP-sialic acid-PEG and 0.1U/mL of CST-II at 32° C. for 2 days. To monitor the incorporation ofsialic acid-PEG, a small aliquot of the reaction hasCMP-SA-PEG-fluorescent ligand added; the label incorporated into thepeptide is separated from the free label by gel filtration on a TosoHaas G3000SW analytical column using PBS buffer (pH 7.1). Thefluorescent label incorporation into the peptide is quantitated using anin-line fluorescent detector. After 2 days, the reaction mixture ispurified using a Toso Haas G3000SW preparative column using PBS buffer(pH 7.1) and collecting fractions based on UV absorption. The product ofthe reaction is analyzed using SDS-PAGE and IEF analysis according tothe procedures and reagents supplied by Invitrogen. Samples of nativeand PEGylated interferon-alpha are dialyzed against water and analyzedby MALDI-TOF MS.

Preparation of Interferon-alpha-(alpha2,6)-Sialyl-PEG. Interferon-alpha,containing only O-linked GalNAc, was dissolved at 2.5 mg/mL in 50 mMTris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH 7.2. The solution is incubatedwith 1 mM CMP-sialic acid-PEG and 0.1 U/mL of ST6GalNAcI or II at 32° C.for 2 days. To monitor the incorporation of sialic acid-PEG, a smallaliquot of the reaction had CMP-SA-PEG-fluorescent ligand added; thelabel incorporated into the peptide is separated from the free label bygel filtration on a Toso Haas G3000SW analytical column using PBS buffer(pH 7.1). The fluorescent label incorporation into the peptide isquantitated using an in-line fluorescent detector. After 2 days, thereaction mixture is purified using a Toso Haas G3000SW preparativecolumn using PBS buffer (pH 7.1) and collecting fractions based on UVabsorption. The product of the reaction is analyzed using SDS-PAGE andIEF analysis according to the procedures and reagents supplied byInvitrogen. Samples of native and PEGylated interferon-alpha aredialyzed against water and analyzed by MALDI-TOF MS.

35. GlycoPEGylation of Interferon-β-1a with PEG (10 kDa) and PEG (20kDa)

This example illustrates a procedure PEGylate Interferon-β with eitherPEG (10 kDa) or PEG (20 kDa).

Briefly, Interferon-β-1a (INF-β) was obtained from Biogen (Avonex™). TheIFN-β was first purified by Superdex-75 chromatography. The IFN-β wasthen desialylated with Vibrio cholerae sialidase. The INF-β was thenPEGylated with SA-PEG (10 kDa) or SA-PEG (20 kDa) and purified withSuperdex-200 chromatography.

Superdex-75 chromatography purification. INF-β (150 μg) was applied to aSuperdex-75 column (Amersham Biosciences, Arlington Heights, Ill.) andeluted with PBS with 0.5 M NaCl, 0.02 Tween-20, 20 mM histidine and 10%glycerol. The eluant was monitored for absorbance at 280 nm (FIGS. 172Aand 172B) and fractions were collected. Peaks 4 and 5 were pooled,concentrated in an Amicon Ultra 15 spin filter (Millipore, Billerica,Mass.), and the buffer was exchanged to TBS with 5 mM CaCl₂, 0.02%Tween-20, 20 mM histidine and 10% glycerol.

Sialidase Reaction. The INF-β was then desialydated with Vibrio cholerasalidase 70 mU/ml, CALBIOCHEM®, EMD Biosciences, Inc., San Diego,Calif.) on agarose in TBS with 5 mM CaCl₂, 0.02% Tween-20, 20 mMhistidine and 10% glycerol. The reaction was carried out at 32° C. for18 hours. The INF-β was removed from the agarose with a 0.22 μm Spin-X™filter (Corning Technology, Inc., Norcross, Ga.). FIG. 173A depicts theMALDI analysis of glycans released from native INF-β. The native INF-βhas many glycoforms containing terminal sialic acid moieties. FIG. 173Bdepicts the MALDI analysis of glycans released from desialylated INF-β.The desialylated INF-β has primarily one glycoform which is bi-antennarywith terminal galactose moieties.

Lectin Dot-Blot Analysis of Sialylation. Samples of the INF-β from thedesialidase reaction were dot-blotted onto nitrocellulose and thenblocked with Tris buffered saline (TBS: 0.05M Tris, 0.15M NaCl, pH 7.5)and DIG kit (glycan differentiation kit available from Roche #1 210 238)blocking buffer. Some of the blots were incubated with Maackia amurensisagglutinin (MAA) labeled with digoxogenin (DIG) (Roche Applied Science,Indianapolis, Ill.) to detect α2,3-sialylation of INF-β. These blotswere washed with TBS then incubated with anti-digitonin antibody labeledwith alkaline phosphatase, then washed again with TBS and developed withNBT/X-phosphate solution, wherein NBT is 4-nitro blue tetrazoliumchloride and X-phosphate is 5-bromo-4-chloro-3indoyl phosphate. The leftside of FIG. 174 depicts the results of the MAA blot of INF-β after thedesialylation reaction. The INF-β is partially disialylated, asindicated by the decrease in dot development as compared to native INF-βin the desialylated samples.

Other blots were incubated with Erthrina cristagalli lectin (ECL)labeled with biotin (Vector Laboratories, Burlingame, Calif.) to detectexposed galactose residues on INF-β. After incubation with 2.5 μg/mlECL, the blots were washed in TBS and incubated with streptavidinlabeled with alkaline phosphatase. The blots were then washed again anddeveloped. The right side of FIG. 174 depicts the ECL blot afterdevelopment. The increased intensity of the dot of desialylated INF-β ascompared to the native INF-β indicate more exposed galactose moietiesand therefore extensive desialylation.

PEGylation of Desialylated INF-β with SA-PEG (10 kDa). DesialylatedINF-β (0.05 mg/ml) was PEGylated with ST3Gal3 (50 mU/ml) and CMP-SA-PEG(10 kDa) (250 μM) in an appropriate buffer of TBS+5 mM CaCl₂, 0.02%Tween 20, 20 mM histidine, 10% glycerol for 50 hours at 32° C. FIG. 175depicts the SDS-PAGE analysis of the reaction products showing PEGylatedINF-β at approximately 98 kDa.

PEGylation of Desialylated INF-β with SA-PEG (20 kDa). DesialylatedINF-β (0.5 mg/ml) was PEGylated with ST3Gal3 (170 mU/ml) and CMP-SA-PEG(20 kDa) in an appropriate buffer of TBS+5 mM CaCl₂, 0.02% Tween 20, 20mM histidine, 10% glycerol for 50 hours at 32° C. FIG. 176 depicts theSDS-PAGE analysis the products of the PEGylation reaction. The PEGylatedINF-β has many higher molecular weight bands not found in the unmodifiedINF-β indicating extensive PEGylation.

Superdex-200 Purification of INF-β PEGylated with PEG (10 kDa). Theproducts of the PEGylation reaction were separated on a Superdex-200column (Amersham Biosciences, Arlington Heights, Ill.) in PBS with 0.5NaCl, 0.02 Tween-20, 20 mM histidine and 10% glycerol at 1 ml/min and 30cm/hr flow. The eluant was monitored for absorbance at 280 nm (FIG. 177)and fractions were collected. Peaks 3 and 4 were pooled and concentratedin an Amicon Ultra 15 spin filter.

Bioassay of INF-β PEGylated with PEG (10 kDa).

The test is inhibition of the proliferation of the lung carcinoma cellline, A549. The A549 cell line are lung carcinoma adherent cells growingin RPMI+10% FBS at 37° C. 5% CO₂. They can be obtained from ATCC #CCL-185. Wash the cells with 10 ml of PBS and remove the PBS. Add 5 mlof trypsin, incubate for 5 minutes at room temperature or 2 minutes at37° C. When the cells are detached resuspend into 25 ml of media andcount the cells. Dilute the cells at a concentration of 10000 cells/mland add 200 ul/well (96 wells plate). Incubate for 4 hours at 37° C. 5%CO₂. Prepare 1 ml of IFN B at a concentration of 0.1 ug/ml. Filter itunder the hood with a 0.2 um filter. Add 100 ul per well (8 replicates=1lane). Incubate for 3 days (do not let the cells go to confluence).Remove 200 ul of media (only 100 ul per well left). Add 25 μl of MTT(Sigma) (5 mg/ml filtered 0.22 μm). Incubate for 4 hours at 37° C. and5% CO₂. Aspirate the media gently and add 100 μl of a mixture ofisopropanol (100 ml and 6N HCl. Aspirate up and down to homogenize thecrystal violet. Read OD 570 nm (remove the background at 630 or 690 nm).

FIG. 178 depicts the results of the bioassay of the peaks containingINF-β PEGylated with PEG (10 kDa) as eluted from the Superdex-200column.

Superdex-200 Purification of INF-β PEGylated with PEG (20 kDa). Theproducts of the PEG (20 kDa) PEGylation reaction were separated on aSuperdex-200 column (Amersham Biosciences, Arlington Heights, Ill.) inPBS with 0.5 NaCl, 0.02 Tween-20, 20 mM histidine and 10% glycerol at 1ml/min flow. The eluant was monitored for absorbance at 280 nm (FIG.179) and fractions were collected. Peak 3 contained most of the INF-βPEGylated with PEG (20 kDa).

Endotoxin test of INF-β PEGylated with PEG (20 kDa).

Limulus Lysate Test was performed, BioWhittaker #50-647 U

TABLE 24 Results of the endotoxin test of INF-β PEGylated with PEG (20kDa). Concentration INF-β with PEG (20 kDa) 10 EU/ml 0.06 mg/ml  0.16EU/μg INF-β with PEG (20 kDa)  1 EU/ml 0.07 mg/ml 0.014 EU/μg NativeINF-β 40 EU/ml  0.1 mg/ml  0.4 EU/μg

Remicade™

36. GlycoPEGylation of Remicade™ Antibody

This example sets forth the procedure to glycoPEGylate a recombinantantibody molecule by introducing PEG molecules to the Fc region glycans.Here Remicade™, a TNF-R:IgG Fc region fusion protein, is the exemplarypeptide.

Preparation of Remicade™-Gal-PEG (10 kDa). Remicade™ is dissolved at 2.5mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 5 mM MnCl₂, 0.05% NaN₃, pH 7.2.The solution is incubated with 1 mM UDP-galactose-PEG (10 kDa) and 0.1U/mL of galactosyltransferase at 32° C. for 2 days to introduce the PEGin the Fc region glycans. To monitor the incorporation of galactose, asmall aliquot of the reaction has ¹⁴C-galactose-UDP ligand added; thelabel incorporated into the peptide is separated from the free label bygel filtration on a Toso Haas G3000SW analytical column using PBS buffer(pH 7.1). The radioactive label incorporation into the peptide isquantitated using an in-line radiation detector.

When the reaction is complete, the reaction mixture is purified using aToso Haas TSK-Gel-3000 preparative column using PBS buffer (pH 7.1) andcollecting fractions based on UV absorption. The fractions containingproduct are combined, concentrated, buffer exchanged and thenfreeze-dried. The product of the reaction is analyzed using SDS-PAGE andIEF analysis according to the procedures and reagents supplied byInvitrogen. Samples are dialyzed against water and analyzed by MALDI-TOFMS.

Rituxan™

37. Glycoconjugation of Geldanamycin to Rituxan™

This example sets forth the glycoconjugation of a small molecule, suchas geldanamycin, to the Fc region glycans of an antibody produced in CHOcells, such as Rituxan™. Here, the antibody Rituxan™ is used, but one ofskill in the art will appreciate that the method can be used with manyother antibodies.

Preparation of Rituxan™-Gal-linker-geldanamycin. Rituxan™ is dissolvedat 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 5 mM MnCl₂, 0.05% NaN₃, pH7.2. The solution is incubated with 1 mMUDP-galactose-linker-geldanamycin and 0.1 U/mL of galactosyltransferaseat 32° C. for 2 days to introduce the geldanamycin in the Fc regionglycans. To monitor the incorporation of galactose, a small aliquot ofthe reaction has ¹⁴C-galactose-UDP ligand added; the label incorporatedinto the peptide is separated from the free label by gel filtration on aToso Haas G3000SW analytical column using PBS buffer (pH 7.1). Theradioactive label incorporation into the peptide is quantitated using anin-line radiation detector.

When the reaction is complete, the reaction mixture is purified using aToso Haas TSK-Gel-3000 preparative column using PBS buffer (pH 7.1) andcollecting fractions based on UV absorption. The fractions containingproduct are combined, concentrated, buffer exchanged and thenfreeze-dried. The product of the reaction is analyzed using SDS-PAGE andIEF analysis according to the procedures and reagents supplied byInvitrogen. Samples are dialyzed against water and analyzed by MALDI-TOFMS.

Rnase

38. Remodeling High Mannose N-glycans to Hybrid and Complex N-glycans:Bovine Pancreatic RNase

This example sets forth the preparation of bovine pancreas RNase withhybrid or complex N-glycans. The high mannose N-linked glycans of theRNase are enzymatically digested and elaborated to create hybridN-linked glycans. Additionally, the high mannose N-linked glycans of theRNase are enzymatically digested and elaborated to create complexN-linked glycans.

High mannose structures of N-linked oligosaccharides in glycopeptidescan be modified to hybrid or complex forms using the combination ofα-mannosidases and glycosyltransferases. This example summarizes theresults in such efforts using a simple N-Glycan as a model substrate.

Ribonuclease B (RNaseB) purified from bovine pancreas (Sigma) is aglycopeptide consisting of 124 amino acid residues. It has a singlepotential N-glycosylation site modified with high mannose structures.Due to its simplicity and low molecular weight (13.7 kDa to 15.5 kDa),ribonuclease B is a good candidate to demonstrate the feasibility of theN-Glycan remodeling from high mannose structures to hybrid or complexN-linked oligosaccharides. The MALDI-TOF spectrum of RNaseB (FIG. 180A)and HPLC profile for the oligosaccharides cleaved from RNaseB byN-Glycanase (FIG. 180B) indicated that, other than a small portion ofthe non-modified peptide, the majority of N-glycosylation sites of thepeptide are modified with high mannose oligosaccharides consisting of 5to 9 mannose residues.

Conversion of high mannose N-Glycans to hybrid N-Glycans. High mannoseN-Glycans were converted to hybrid N-Glycans using the combination of(α1,2-mannosidase, GlcNAcT-I (β-1,2-N-acetyl glucosaminyl transferase),GalT-I (β1,4-galactosyltransfease) and α2,3-sialyltransferase/orα2,6-sialyltransferase as shown in FIG. 181.

As an example, high mannose structures in RNaseB were successfullyconverted to hybrid structures.

Man₅GlcNAc₂—R was obtained from Man₅₋₉GlcNAc₂—R catalyzed by a singleα1,2-mannosidase cloned from Trichoderma reesei (FIG. 182). RNase B (1g, about 67 μmol) was incubated at 30° C. for 45 hr with 15 mU of therecombinant T. reesei α1,2-mannosidase in MES buffer (50 mM, pH 6.5) ina total volume of 10 mL. Man₆₋₉GlcNAc₂-protein structures have beensuccessfully converted to Man₅GlcNAc₂-protein with high efficiency bythe recombinant mannosidase.

Alternately, Man₅GlcNAc₂—R was obtained from Man₅₋₉GlcNAc₂—R catalyzedby a single α1,2-mannosidase purified from Aspergillus saitoi (FIG.183). RNase B (40 μg, about 2.7 nmol) was incubated at 37° C. for 42.5hr with 25 μU of the commercial A. saitoi α1,2-mannosidase (Glyko orCalBioChem) in NaOAC buffer (100 mM, pH 5.0) in a total volume of 20 μl.Man₆₋₉GlcNAc₂-protein structures were successfully converted toMan₅GlcNAc₂-protein by the commercially available mannosidase. However,a new peak corresponding to the GlcNAc-protein appears in the spectrum,indicating the possible contamination of endoglycosidase H in thepreparation. Although several mammalian alpha-mannosidases were requiredto achieve this step, the fungal α1,2-mannosidase was very efficient toremove all α1,2-linked mannose residues.

GlcNAcT-I then added a GlcNAc residue to the Man₅GlcNAc₂—R (FIG. 184).The reaction mixture after the T. reesei α1,2-mannosidase reactioncontaining RNase B (600 μg, about 40 nmol) was incubated withnon-purified recombinant GlcNAcT-I (34 mU) in MES buffer (50 mM, pH 6.5)containing MnCl₂ (20 mM) and UDP-GlcNAc (5 mM) in a total volume of 400μl. at 37° C. for 42 hr. A GlcNAc residue was quantitatively added toMan₅GlcNAc₂-protein by the recombinant GlcNAcT-I.

A Gal residue was then added using GalT 1 (FIG. 185). The reactionmixture after the GnT-I reaction containing RNase B (120 μg, about 8nmol) was incubated at 37° C. for 20 hr with 3.3 mU of the recombinantGalT-1 in Tris-HCl buffer (100 mM, pH 7.3) containing UDP-Gal (7.5 mM)and MnCl₂ (20 mM) in a total volume of 100 μl. A Gal residue was addedto about 98% of the GlcNAc-Man₅GlcNAc₂-protein by the recombinant GalT1.

The next step was the addition of a sialic acid using anα2,3-sialyltransferase or an α2,6-sialyltransferase (FIG. 186). As anexample, ST3Gal III, an α2,3-sialyltransferase was used. The reactionmixture after the GalT-1 reaction containing RNase B (13 μg, about 0.87nmol) was incubated at 37° C. for 16 hr with 8.9 mU of recombinantST3Gal III in Tris-HCl buffer (100 mM, pH 7.3) containing CMP-Sialicacid (5 mM) and MnCl₂ (20 mM) in a total volume of 20 μl. A sialic acidresidue was added to about 90% of the Gal-GlcNAc-Man₅GlcNAc₂-protein byrecombinant ST3Gal III using CMP-SA as the donor. The yield can befurther improved by adjusting the reaction conditions.

For convenience, no purification or dialysis step was required aftereach reaction described above. More interesting, GalT 1 and ST3Gal IIIcan be combined in a one-pot reaction. Similar yields were obtained ascompared with the separate reactions. The reaction mixture after theGlcNAcT-I reaction containing RNase B (60 μg, about 4 nmol) wasincubated at 37° C. for 20 hr with 1.7 mU of recombinant GalT 1, 9.8 mUof recombinant ST3 Gal III in Tris-HCl buffer (100 mM, pH 7.3)containing UDP-Gal (7.5 mM), CMP-sialic acid (5 mM) and MnCl₂ (20 mM) ina total volume of 60 μl.

As shown in FIG. 187, SA-PEG (10 kDa) was successfully added to theRNaseB. The reaction mixture after the GalT-1 reaction containing RNaseB (6.7 μg, about 0.45 nmol) was dialyzed against H₂O for 1 hour at roomtemperature and incubated at 37° C. for 15.5 hours with 55 mU of therecombinant ST3Gal III in Tris-HCl buffer (50 mM, pH 7.3) containingCMP-SA-PEG (10 kDa) (0.25 mM) and MnCl₂ (20 mM) in a total volume of 20μl. PEG-modified sialic acid residues were successfully added to theGal-GlcNAc-Man₅GlcNAc₂-peptide by the recombinant ST3Gal III. The yieldcan be further improved by adjusting the reaction conditions.

Conversion of high mannose N-Glycans to complex N-Glycans. To achievethis conversion, a GlcNAcβ1,2Man₃GlcNAc₂-peptide intermediate isobtained. As shown in FIG. 188, there are at least four feasible routesto carry out the reaction from Man₅GlcNAc₂-peptide to this intermediate:

Route I: The Man₅GlcNAc₂-peptide produced by the fungal α1,2 mannosidaseis a substrate of GlcNAc transferase I (GlcNAcT-I, enzyme 2) which addsone GlcNAc. The terminal α1,3- and α1,6-linked mannose residues ofGlcNAcMan₅GlcNAc₂-peptide is removed by Golgi α-mannosidase II (ManII,enzyme 5). This route is a part of the natural pathway for theprocessing of N-linked oligosaccharides carried out in higher organisms.

Route II: Two mannose residues are first removed by an α-mannosidase(enzyme 6), then a GlcNAc is added by GlcNAcT-I (enzyme 2). Other thanits natural acceptor Man₅GlcNAc₂—R, GlcNAcT-I can also recognizeMan₃GlcNAc₂—R as its substrate and add one GlcNAc to the mannose corestructure to form GlcNAcMan₃GlcNAc₂-peptide.

Route III: The α1,6-linked mannose is removed by an α1,6-mannosidase,followed y the addition of GlcNAc by GlcNAcT-I and removal of theterminal α1,3-linked mannose by an α1,3-mannosidase. From theexperimental data obtained, GlcNAcT-I can recognize thisMan₄GlcNAc₂-peptide as acceptor and add one GlcNAc residue to formGlcNAcMan₄GlcNAc₂-peptide.

Route IV: Similar to Route III, α1,3-linked mannose is removed by anα1,3-mannosidase, followed by GlcNAcT-I reaction. Then the terminalα1,6-linked mannose can be removed by an α1,6-mannosidase.

After the function of GlcNAcT-I (responsible for the addition of theGlcNAc β1,2-linked to the α1,3-mannose on the mannose core) andGlcNAcT-II (responsible for the addition of a second GlcNAc β1,2-linkedto the α1,6-mannose on the mannose core), the GlcNAc₂Man₃GlcNAc₂-peptidecan be processed by GalT 1 and sialyltransferase to form bi-antennarycomplex N-Glycans. Other GlcNAc transferases such as GlcNAcT-IV,GlcNAcT-V, and/or GlcNAcT-VI (FIG. 188 and FIG. 189) can alsoglycosylate the GlcNAc₂Man₃GlcNAc₂-peptide. Additional glycosylation bythe GalT 1 and sialyltransferases will form multi-antennary complexN-glycans. The enzyme GlcNAcT-III catalyzes the insertion of a bisectingGlcNAc, thus preventing the actions of ManII and subsequent action oftransferases GlcNAcT-II, GlcNAcT-IV and GlcNAcT-V.

Tissue-Type Plasminogen Activator (TPA)

39. Fucosylation of TPA to Create Sialyl Lewis X

This example sets forth the preparation of Tissue Tissue-typePlasminogen Activator (TPA) with N-linked sialyl Lewis X antigen.

Sialylation. TPA expressed in mammalian cells will often contain amajority of the glycans terminating in sialic acid, but to ensurecomplete sialylation, it would be beneficial to first perform an invitro sialylation. TPA in a suitable buffer (most preferably between pH5.5 and 9, for example Tris buffered saline, pH 7.2) is incubated withCMP sialic acid and sialyltransferase for a time sufficient to convertany glycans lacking sialic acid to sialylated species. Typicalconditions would be 1 mg/mL TPA, 3 mM CMP sialic acid, 0.02 U/mLST3Gal3, 32° C. for 24 hours. Microbial growth can be halted either bysterile filtration or the inclusion of 0.02% sodium azide. The TPAconcentration is most preferably in the range 0.1 mg/mL up to thesolubility limit of the peptide. The concentration of CMP-SA should besufficient for there to be excess over the available sites, and mightrange from 50 μM up to 50 mM, and the temperature from 2° C. up to 40°C. The time required for complete reaction will depend on thetemperature, the relative amounts of enzyme to acceptor substrate, thedonor substrate concentration, and the pH. Other sialyltransferases thatmay be capable of adding sialic acid in 2,3 linkage include ST3Gal4;microbial transferases could also be used.

Fucosylation. Typical conditions for fucosylation would be 1 mg/mL TPA,3 mM GDP-fucose, 0.02 U/mL FTVI, 5 mM MnCl₂, 32° C. for 24 H in Trisbuffered saline. Microbial growth can be halted either by sterilefiltration or the inclusion of 0.02% sodium azide. The TPA concentrationis most preferably in the range 0.1 mg/mL up to the solubility limit ofthe peptide. The concentration of GDP-fucose should be sufficient forthere to be excess over the available sites, and might range from 50 μMup to 50 mM, and the temperature from 2° C. up to 40° C. The timerequired for complete reaction will depend on the temperature, therelative amounts of enzyme to acceptor substrate, the donor substrateconcentration, and the pH. Other fucosyltransferases that may be capableof making sialyl Lewis x include FTVII, FTV, FTIII, as well as microbialtransferases could also be used.

40. Trimming of High Mannose to Tri-Mannose Core Structure: Tissue-TypePlasminogen Activator Produced in CHO

This example sets forth the preparation of Tissue-type PlasminogenActivator with a trimannose core by trimming back from a high mannoseglycan.

Tissue-type plasminogen activator (TPA) is currently produced in ChineseHamster Ovary (CHO) cells and contains a low amount of high mannoseN-linked oligosaccharide. The mannoses can be trimmed down using avariety of the specific mannosidases. The first step is to generateMan5GlcNAc2(Fuc0-1) from Man9GlcNAc2(Fuc0-1). This can be done usingmannosidase I. Then either GlcNAcT1 (GlcNAc transferase I) is used tomake GlcNAc1Man5GlcNAc2(Fuc0-1) or Mannosidase III is used to makeMan3GlcNAc2(Fuc0-1). From Man3GlcNAc2(Fuc0-1),GlcNAc1Man3GlcNAc2(Fuc0-1) can be produced using GlcNAcT1 or fromGlcNAc1Man5GlcNAc2(Fuc0-1), GlcNAc1Man3GlcNAc2(Fuc0-1) can be producedusing Mannosidase II. GlcNAc1Man3GlcNAc2(Fuc0-1) is then converted intoGlcNAc2Man3GlcNAc2(Fuc0-1) using GlcNAcTransferase II (GlcNAcTII). Thetwo terminal GlcNAc residues are then galactosylated using GalTI andthen sialylated with SA-PEG using ST3GalIII.

Conversely, TPA can be produce in yeast or fungal systems. Similarprocessing would be required for fungal derived material.

41. Generation and PEGylation of GlcNAc-ASN Structures: TPA Produced inYeast

This example sets forth the preparation of PEGylated GlcNAc-Asnstructures on a peptide such as TPA expressed in yeast.

Yeast expression is expected to result in a TPA which contains a singleN-linked mannan-type structure. This recombinant glycoprotein is firsttreated with endoglycosidase H to generate GlcNAc structures on theasparagine (Asn) residues on the peptide.

The GlcNAc-Asn structures on the peptide/protein backbone are then bemodified with galactose or galactose-PEG using UDP-galactose orUDP-galactose-6-PEG, respectively, and a galactosyltransferase such asGalT1. In one case, the galactose-PEG is the terminal residue. In thesecond case, the galactose is further modified with SA-PEG using aCMP-SA-PEG donor and a sialyltransferase such as ST3GalIII. In anotherembodiment, the GlcNAc-Asn structures on the peptide/protein backbonemay be galactosylated and sialylated as described above, and thenfurther sialylated using CMP-SA-PEG and an α2,8-sialyltransferase suchas the enzyme encoded by the Campylobacter jejuni cst-II gene.

Transferrin

42. GlycoPEGylation of Transferrin

This example sets forth the preparation of asialotransferrin and itssialylation with PEG-CMP-sialic acid.

Preparation of Asialo-transferrin. Human-derived holo-Transferrin, (10mg) was dissolved in 500 μL of 50 mM NaOAc, 5 mM CaCl₂, pH 5.5. To thissolution was added 500 mU Neuraminidase II (Vibrio cholerae) and thereaction mixture was shaken gently for 20.5 hours at 37° C. The reactionmixture was added to the prewashed N-(p-aminophenyl)oxamic acid-agaroseconjugate (600 μL) and the washed beads gently rotated for 24 hours at4° C. The mixture was centrifuged at 10,000 rpm and the supernatant wascollected. The reaction mixture was adjusted to 5 mM EDTA by addition of100 μL of 30 mM EDTA to the washed beads, which were gently rotated for20 hours at 4° C. The suspension was centrifuged for 2 minutes at 10,000rpm and the supernatant was collected. The beads were washed 5 timeswith 0.35 mL of 50 mM NaOAc, 5 mM CaCl₂, 5 mM EDTA, pH 5.5 and allsupernatants were pooled. The enzyme solution was dialyzed twice at 4°C. into 15 mM Tris-HCl, 1 M NaCl, pH 7.4. 0.3 mL of the transferrinsolution (3.3 mL total) was removed and dialyzed twice against water.The remainder was dialyzed twice more at 4° C. against phosphatebuffered saline. The dialyzed solution was stored at −20° C. Proteinsamples were analyzed by IEF Electrophoresis. Samples (9 μL, 25 μg) werediluted with 16 μL Tris buffer and mixed with 25 μL of the sampleloading buffer and applied to Isoelectric Focusing Gels (pH 3-7). Gelswere run and fixed using standard procedures. Gels were stained withColloidal Blue Stain.

Sialyl-PEGylation of asialo-Transferrin. Desialylated transferrin (250μg) and CMP-sialic acid or CMP-SA-PEG (1 kDa or 10 kDa)(0.05 μmol) weredissolved in 69 μL 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH 7.2 in1.5 mL plastic tubes. The tubes were vortexed briefly and 100 mU ST3Gal3(90 μL) were added (total volume 250 μL). The tubes were vortexed againand mixed gently for 24 hours at 32° C. The reactions were stopped byfreezing at −80° C. Novex Tris-Glycine 8-16% 1 mm gels were used for SDSPAGE analysis (FIG. 190). Samples (25 μL, 25 μg) were mixed with 25 μLof sample loading buffer and 0.4 μL of β-mercaptoethanol and heated for6 minutes at 85° C. Gels were run using standard conditions and stainedwith Colloidal Blue Stain. IEF gels were also performed as describedabove FIG. 191). Samples were also dialyzed against water analyzed byMALDI-TOF.

Results. MALDI was also performed. Native transferrin (78729);asialotransferrin (78197); resialylated transferrin (79626/80703); withSA-PEG 1k (79037 (1); 80961 (2); 82535 (3); 84778 (4)); with SA-PEG 5 k(90003 (2); 96117 (3); 96117 (4)); with SA-PEG 10 k (100336 (2); 111421(3); 122510 (4)).

43. Transferrin-GDNF

This example sets forth the procedures for the glycoconjugation ofproteins, and in particular, transferrin is glycoconjugated to GDNF.Transferrin-SA-Linker-Gal-UDP is prepared from transferrin. Thegalactose residue is removed from GNDF glycans, andTransferrin-SA-Linker-Gal-UDP is conjugated to GNDF glycans using agalactosyltransferase.

Preparation of agalacto-GDNF. GDNF produced in NSO cells (NSO murinemyeloma cells) is dissolved at 2.5 mg/mL in 50 mM Tris 50 mM Tris-HCl pH7.4, 0.15 M NaCl, and is incubated with 300 mU/mLbeta-galactosidase-agarose conjugate for 16 hours at 32° C. To monitorthe reaction a small aliquot of the reaction is diluted with theappropriate buffer and a IEF gel performed according to Invitrogenprocedures. The mixture is centrifuged at 10,000 rpm and the supernatantis collected. The supernatant is dialyzed at 4 ° C. against 50 mMTris-HCl pH 7.4, 1 M NaCl, 0.05% NaN₃ and then twice more against 50 mMTris-HCl pH 7.4, 1 M NaCl, 0.05% NaN₃. The dialyzed solution is thenconcentrated using a Centricon Plus 20 centrifugal filter and stored at−20° C. The conditions for the IEF gel are run according to theprocedures and reagents provided by Invitrogen. Samples are dialyzedagainst water and analyzed by MALDI-TOF MS.

Preparation of Transferrin-SA-Linker-Gal-UDP. Asialo-transferrin isdissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH7.2. The solution is incubated with CMP-sialic acid-linker-Gal-UDP(molar amount to add 1 molar equivalent of nucleotide sugar totransferrin) and 0.1 U/mL of ST3Gal3 at 32° C. for 2 days. To monitorthe incorporation of sialic acid, a small aliquot of the reaction has¹⁴C-SA-UDP ligand added; the label incorporated into the peptide isseparated from the free label by gel filtration on a Toso Haas G3000SWanalytical column using PBS buffer (pH 7.1). The radioactive labelincorporation into the peptide is quantitated using an in-line radiationdetector.

The solution is incubated with 5 mM CMP-sialic acid and 0.1 U/mL ofST3Gal3 (to cap any unreacted transferrin glycans) at 32° C. for 2 days.The incorporation into the peptide is quantitated using an in-line UVdetector. After 2 days, the reaction mixture is purified using a TosoHaas G3000SW preparative column using PBS buffer (pH 7.1) and collectingfractions based on UV absorption. The product of the reaction isanalyzed using SDS-PAGE and IEF analysis according to the procedures andreagents supplied by Invitrogen. Samples are dialyzed against water andanalyzed by MALDI-TOF MS.

Preparation of Transferrin-SA-Linker-Gal-GDNF. Thetransferrin-SA-Linker-Gal-UDP prepared as described above is dissolvedat 2.5 mg/ml in 50 mM Tris-HCl, 0.15 M NaCl, 5 mM MnCl₂, 0.05% NaN₃, pH7.2. The solution is incubated with 2.5 mg/mL agalacto-GDNF and 0.1 U/mLof galactosyltransferase at 32° C. for 2 days. To monitor theincorporation of galactose, a small aliquot of the reaction has¹⁴C-galactose-UDP ligand added; the label incorporated into the peptideis separated from the free label by gel filtration on a Toso HaasG3000SW analytical column using PBS buffer (pH 7.1). The radioactivelabel incorporation into the peptide is quantitated using an in-lineradiation detector.

When the reaction is complete, the solution is incubated with 5 mMUDP-Gal and 0.1 U/mL of galactosyltransferase (to cap any unreactedtransferrin glycans) at 32° C. for 2 days followed by addition of 5 mMCMP-SA and 0.1 U/mL of ST3Gal3. After 2 additional days, the reactionmixture is purified using a Toso Haas G3000SW preparative column usingPBS buffer (pH 7.1) collecting fractions based on UV absorption. Theproduct of the reaction is analyzed using SDS-PAGE and IEF analysisaccording to the procedures and reagents supplied by Invitrogen. Samplesare dialyzed against water and analyzed by MALDI-TOF MS.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A glycoPEGylated EPO peptide comprising an EPO peptide and at leastone glycan and at least one poly(ethylene glycol) molecule covalentlyattached to said glycan, wherein said glycoPEGylated EPO peptide is madeby a method comprising: adding said poly(ethylene glycol) molecule tosaid EPO peptide using a glycosyltransferase.
 2. The glycoPEGylated EPOpeptide of claim 1, comprising at least one mono-antennary glycan. 3.The glycoPEGylated EPO peptide of claim 1, wherein each of said at leastone glycan is N-linked and mono-antennary.
 4. The glycoPEGylated EPOpeptide of claim 1, wherein each of said at least one glycan is N-linkedand at least one of said glycans comprise said poly(ethylene glycol). 5.The glycoPEGylated EPO peptide of claim 4, wherein more than one of saidglycans comprises said poly(ethylene glycol).
 6. The glycoPEGylated EPOpeptide of claim 1, wherein each of said at least one glycan is N-linkedand all of said glycans comprise said poly(ethylene glycol).
 7. TheglycoPEGylated EPO peptide of claim 1, comprising at least threemono-antennary glycans having said poly(ethylene glycol) covalentlyattached thereto.
 8. The glycoPEGylated EPO peptide of claim 1 whereinsaid poly(ethylene glycol) is linked to at least one sugar moietyselected from the group consisting of fucose (Fuc), N-acetylglucosamine(GIcNAc), galactose (Gal) and a sialic acid (SA).
 9. The glycoPEGylatedEPO peptide of claim 8, wherein said sialic acid is N-acetylneuraminicacid.
 10. The glycoPEGylated EPO peptide of claim 1, wherein said EPOpeptide does not comprise an O-linked glycan.
 11. The glycoPEGylated EPOpeptide of claim 1 wherein said EPO peptide comprises at least oneO-linked glycan.
 12. The glycoPEGylated EPO peptide of claim 11, whereinsaid O-linked glycan comprises said poly(ethylene glycol) covalentlyattached thereto.
 13. The glycoPEGylated EPO peptide of claim 12,wherein said EPO peptide is recombinantly expressed in a cell.
 14. TheglycoPEGylated EPO peptide of claim 13, wherein said cell is selectedfrom the group consisting of an insect cell, a fungal cell and amammalian cell.
 15. The glycoPEGylated EPO peptide of claim 14, whereinsaid cell is an insect cell.
 16. The glycoPEGylated EPO peptide of claim14, wherein said cell is a yeast cell.
 17. The glycoPEGylated EPOpeptide of claim 14, wherein said cell is a mammalian cell.
 18. TheglycoPEGylated EPO peptide of claim 17, wherein said mammalian cell is aCHO cell.
 19. The glycoPEGylated EPO peptide of claim 1, wherein saidpoly(ethylene glycol) has a molecular weight selected from the groupconsisting of about 1 kDa, 2 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa and 40kDa.
 20. The glycoPEGylated EPO peptide of claim 19, wherein saidpoly(ethylene glycol) has a molecular weight of 20 kDa.
 21. TheglycoPEGylated EPO peptide of claim 1, wherein said EPO peptide isselected from the group consisting of a naturally occurring EPO peptideand a mutated EPO peptide.
 22. The glycoPEGylated EPO peptide of claim21, wherein said mutated EPO peptide comprises the amino acid sequenceof SEQ ID NO:73 having at least one mutation selected from the groupconsisting of Arg¹³⁹ to Ala¹³⁹, Arg¹⁴³ to Ala¹⁴³ and Lys¹⁵⁴ to Ala¹⁵⁴.23. A method of making a glycoPEGylated EPO peptide, said methodcomprising the step of: (a) contacting an EPO peptide with a mixturecomprising a nucleotide sugar covalently linked to poly(ethylene glycol)and a glycosyltransferase under conditions sufficient to transfer saidpoly(ethylene glycol) to said EPO peptide.
 24. The method of claim 23,wherein the sugar of said nucleotide sugar is selected from the groupconsisting of fucose (Fuc), N-acetylglucosamine (GIcNAc), galactose(Gal) and a sialic acid (SA).
 25. The method of claim 24, wherein saidsialic acid is N-acetylneuraminic acid (NAN).
 26. The method of claim23, wherein said poly(ethylene glycol) has a molecular weight selectedfrom the group consisting of about 1 kDa, 2 kDa, 5 kDa, 10 kDa, 20 kDa,30 kDa and 40 kDa.
 27. The method of claim 26, wherein saidpoly(ethylene glycol) has a molecular weight of 20 kDa.
 28. The methodof claim 23, wherein said EPO peptide is recombinantly expressed in acell.
 29. The method of claim 28, wherein said cell is selected from thegroup consisting of an insect cell, a fungal cell and a mammalian cell.30. The method of claim 29, wherein said cell is an insect cell.
 31. Themethod of claim 29, wherein said cell is a yeast cell.
 32. The method ofclaim 29, wherein said cell is a mammalian cell.
 33. The method of claim32, wherein said mammalian cell is a CHO cell.
 34. The method of claim23, wherein said EPO peptide is selected from the group consisting of anaturally occurring EPO peptide and a mutated EPO peptide.
 35. Themethod of claim 34, wherein said mature EPO peptide has the sequence ofSEQ ID NO:73.
 36. The method of claim 34, wherein said mutated EPOpeptide comprises the amino acid sequence of SEQ ID NO: 73 having atleast one mutation selected from the group consisting of Arg¹³⁹ toAla¹³⁹, Arg¹⁴³ to Ala¹⁴³ and Lys¹⁵⁴ to Ala¹⁵⁴.
 37. The method of claim23, wherein before step (a): (b) contacting said EPO peptide with amixture comprising a nucleotide-N-acetylglucosamine (GlcNAc) moleculeand an N-acetylglucosamine transferase (GnT) for which thenucleotide-GIcNAc is a substrate under conditions sufficient to form abond between said GlcNAc and said EPO, wherein said GnT is selected fromthe group consisting of GnT I, GnT II, GnT III, GnT IV, GnT V and GnTVI.
 38. The method of claim 37, wherein said mixture comprises one GnTselected from the group consisting of GnT I, GnT II, GnT IV, GnT V andGnT VI.
 39. The method of claim 38, wherein said GnT is GnT I.
 40. Themethod of claim 38, wherein said GnT is GnT II.
 41. The method of claim23, wherein said glycoPEGylated EPO peptide comprises at least onemono-anten nary glycan.
 42. The method of claim 23, wherein the sugar ofsaid nucleotide sugar is galactose and said glycosyltransferase isgalactosyl transferase I (GalT I).
 43. The method of claim 37, whereinbefore step (a) but after step (b): (c) contacting said EPO peptide witha mixture comprising a nucleotide galactose (Gal) and galactosyltransferase I (GalT I) under conditions sufficient to transfer galactoseto said EPO peptide.
 44. The method of claim 23, wherein in step (a),the sugar of said nucleotide sugar is sialic acid and saidglycosyltransferase is a sialyltransferase.
 45. The method of claim 44,wherein said sialic acid is N-acetylneuraminic acid (NAN).
 46. Themethod of claim 44, wherein said sialyltransferase is selected from thegroup consisting of α(2,3)sialyltransferase, α(2,6)sialyltransferase and(2,8)sialyltransferase.
 47. A glycoPEGylated EPO peptide made by themethod of claim
 23. 48. A glycoPEGylated EPO peptide, said EPO peptidecomprising the sequence of SEQ ID NO:73 and further comprising an intactglycosyl linking group linking said EPO peptide and a PEG moiety of saidglycoPEGylated EPO peptide.
 49. A glycoPEGylated EPO peptide, said EPOpeptide comprising the sequence of SEQ ID NO:73 and further comprising amutation in said sequence.
 50. A method of making a glycoPEGylated EPOpeptide, said method comprising the steps of: (a) contacting an EPOpeptide with a mixture comprising a nucleotide sugar covalently linkedto poly(ethylene glycol) and a glycosyltransferase under conditionssufficient to transfer said poly(ethylene glycol) to said EPO peptide,wherein said glycosyltransferase is a fucosyltransferase.
 51. The methodof claim 50, wherein said fucosyltransferase is selected from the groupconsisting of fucosyltransferase I, fucosyltransferase III,fucosyltransferase IV, fucosyltransferase V, fucosyltransferase VI andfucosyltransferase VII.
 52. A glycoPEGylated EPO peptide made by themethod of claim
 50. 53. The method of claim 50, wherein said EPO peptideis expressed in a CHO cell.
 54. A method of providing an EPO peptide toa mammal, said method comprising administering an effective amount of aglycoPEGylated EPO peptide comprising an EPO peptide and at least oneglycan and at least one poly(ethylene glycol) molecule covalentlyattached to said glycan, wherein said poly(ethylene glycol) molecule isadded to said EPO peptide using a glycosyltransferase, wherein said EPOpeptide is administered in an amount effective to increase thehematocrit level in said mammal.
 55. The method of claim 54, whereinsaid mammal is a human.
 56. A method of treating a mammal having anemia,said method comprising administering to said mammal a glycoPEGylated EPOpeptide comprising an EPO peptide and at least one glycan and at leastone poly(ethylene glycol) molecule covalently attached to said glycan,wherein said poly(ethylene glycol) molecule is added to said EPO peptideusing a glycosyltransferase, wherein said EPO peptide is administered inan amount effective to increase the hematocrit level in said mammal. 57.The method of claim 56, wherein said mammal is a human.
 58. The methodof claim 57, wherein said anemia is associated with chemotherapy. 59.The glycoPEGylated EPO peptide of claim 1, wherein said at least oneglycan is a biantennary glycan.
 60. The glycoPEGylated EPO peptide ofclaim 1, wherein said at least one glycan is a triantennary glycan. 61.The glycoPEGylated EPO peptide of claim 1, wherein said at least oneglycan is at least a triantennary glycan.
 62. The glycoPEGylated EPOpeptide of claim 1, wherein said at least one glycan comprises at leasttwo glycans selected from mono- and multiantennary glycans.
 63. TheglycoPEGylated EPO peptide of claim 1, wherein said at least one glycanis selected from N-linked glycans and O-linked glycans.
 64. TheglycoPEGylated EPO peptide of claim 1, wherein said at least one glycanis at least two glycans selected from N-linked and O-linked glycans. 65.The glycoPEGylated EPO peptide of claim 1, wherein said EPO peptide hasthe sequence of SEQ ID NO:73.
 66. The glycoPEGylated EPO peptide ofclaim 1, wherein said method comprises the step of: (a) contacting anEPO peptide with a mixture comprising a nucleotide sugar derivatizedwith poly(ethylene glycol) and a glycosyltransferase under conditionssufficient to transfer said poly(ethylene glycol) to said EPO peptide.67. The glycoPEGylated EPO peptide of claim 66, wherein said nucleotidesugar comprises a sugar moiety selected from the group consisting offucose (Fuc), N-acetylglucosamine (GIcNAc), galactose (Gal) and sialicacid (SA).
 68. The glycoPEGylated EPO peptide of claim 67, wherein saidsialic acid is N-acetylneuraminic acid (NAN).
 69. The glycoPEGylated EPOpeptide of claim 66, wherein said method further comprises prior to step(a): (b) contacting said EPO peptide with a mixture comprising anucleotide-N-acetylglucosamine (GIcNAc) molecule and anN-acetylglucosamine transferase (GnT) for which the nucleotide-GIcNAc isa substrate under conditions sufficient to form a bond between saidGIcNAc and said EPO, wherein said GnT is selected from the groupconsisting of GnT I, GnT II, GnT III, GnT IV, GnT V and GnT VI.
 70. TheglycoPEGylated EPO peptide of claim 69, wherein said mixture comprisesone GnT selected from the group consisting of GnT I, GnT II, GnT IV, GnTV and GnT VI.
 71. The glycoPEGylated EPO peptide of claim 70, whereinsaid GnT is GnT I.
 72. The glycoPEGylated EPO peptide of claim 70,wherein said GnT is GnT II.
 73. The glycoPEGylated EPO peptide of claim66, wherein said sugar of said nucleotide sugar is galactose and saidglycosyltransferase is galactosyl transferase I (GaIT I).
 74. TheglycoPEGylated EPO peptide of claim 66, further comprising prior to step(a) but after step (b): (c) contacting said EPO peptide with a mixturecomprising a nucleotide galactose (Gal) and galactosyl transferase I(GaIT I) under conditions sufficient to transfer galactose to said EPOpeptide.
 75. The glycoPEGylated EPO peptide of claim 66, wherein saidnucleotide sugar of step (a) comprises sialic acid and saidglycosyltransferase is a sialyltransferase.
 76. The glycoPEGylated EPOpeptide of claim 75, wherein said sialic acid is N-acetylneuraminic acid(NAN).
 77. The glycoPEGylated EPO peptide of claim 75, wherein saidsialyltransferase is selected from the group consisting ofα(2,3)sialyltransferase, α(2,6)sialyltransferase and(2,8)sialyltransferase.